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To characterize the dosimetric impact of using different sizes of spherical applicators in Intrabeam™ Intraoperative Radiation Therapy (IB-IORT) to treat unicentric cancer lesions after breast-conserving lumpectomy.Using the commissioned depth dose rates, the three-dimensional dosages of the 3.5, 4.0, 4.5, and 5.0 cm spherical applicators of the IB-IORT PRS400 machine were established. Five different cancer cell infiltration depths of unicentric breast lesions were formulated by a linearly declining cancer cell density distribution from the surgical surface. The equivalent uniform dose (EUD), which is the dosage of a homogeneous dose treatment for killing the same amount of cancer cells as IB-IORT in the same target volume, was then calculated using the modified linear quadratic model (MLQ). The radiobiological response of two types of cancer cell lines and three types of normal tissues in the TARGIT-A clinical trial of 20 Gy dose was estimated. The study was carried out for an acutely responding breast cancer cell line with an α/β ratio of 10 and a slow responding breast cancer cell line with an α/β ratio of 3.85, respectively. The cancer cell density at the surgical excision surface was assumed to be 0.01%, 0.1%, 1%, and 10%, respectively. The three types of normal tissue are radiosensitive, moderate radiosensitive, and radioresistant, respectively. The therapeutic ratio (TR), which was defined by a ratio between the survival fractions of normal tissue cells respectively in IB-IORT and in homogeneous dose treatments, was calculated.The EUDs are moderately dependent on the applicator size (increasing from 1 to 10% depending on the cancer infiltrating depth when increasing diameter by 0.5 cm), not on the cancer cell radiosensitivity (differing by less than 1.30% between two cancer cell lines), and not dependent on the cancer cell population density at the surgical excision surface (differing by 0% among the tested surface densities). The EUDs decrease with the cancer cell maximum infiltrating depth. EUD of a 10 mm spherical shell target volume is about 50% of EUD of a 1 mm spherical shell. TRs are dependent on the applicator size, cancer cell infiltrating depth, and the radiosensitivities of cancer cells and normal tissue. A smaller size of applicator was found to produce a greater TR for a shallowly seated lesion (<5 mm), while a large size of applicator is better to treating a deeply seated lesion (>5 mm).The applicator size has a moderate impact on the EUDs, a greater size of applicator will deliver a greater EUD at the same cancer cell infiltrating depth, but a smaller size of applicator is seen to produce a greater TR for the shallow lesions (depth <5 mm). In addition, a greater size of applicator is preferred for treating a deeply seated lesion (depth >5 mm) because it produces a greater TR and EUD than a smaller size of applicator. IB-IORT is a preferable alternative to uniform dose treatment when treating unicentric breast cancer at smaller infiltration depths.