BACKGROUND: Direct cortical stimulation (DCS) is considered the gold-standard for identification of functional brain regions during awake craniotomy for tumor resection. This technique allows maximal resection while preserving neurological function and quality of life. To date, no intra-operative methodology for mapping neural networks, which are crucial for adequate neurological functions, has been described. We describe the feasibility of a novel intra-operative technique, aiming to map critical language sub-networks using simultaneous, multi-site cortical stimulations. METHODS: 15 eligible patients who underwent awake craniotomy for removal of tumors involving language areas participated in this study. Following standard DCS using a bipolar Ojemann stimulator, a strip electrode was placed on “silent” cortex (where DCS has not elicited any language dysfunction). Functional testing was continued while applying simultaneous two to three-site bipolar stimulation via two pairs of adjacent contacts and Ojemann stimulator. Dysfunction was documented when occurred consistently during stimulation. RESULTS: 12 of the 15 patients (80%) experienced language dysfunction following standard single-site stimulation. Two-site simultaneous stimulations (neither causing dysfunction by itself) were associated with additive language dysfunction in 10 of the 15 patients. Three-site stimulations caused additive language dysfunction in 8 patients, as compared to the single- and two- site stimulations. Language dysfunctions caused by multifocal stimulation varied among patients and tumor locations and included primary production and comprehension dysfunctions (i.e. speech arrest and word-deafness, respectively); as well as secondary language dysfunctions such as phonological and semantic paraphasias. Importantly, using multifocal stimulations we identified eloquent regions in patients who did not experience any dysfunction using the standard DCS (two patients). Functional regions identified were spared during surgery. In none of the patients a new neurological deficit was observed following surgery, and in six patients who experienced dysfunction caused by multifocal stimulation, post-operative language function has improved. CONCLUSIONS: In this preliminary study we show that multi-site intra-operative stimulations enables identification of additional functional nodes and sub-networks which are more refined and cannot be mapped with standard single site stimulations. Using such technique may allow for preservation of neural networks and further enhance functional preservation in patients who undergo surgery for brain tumors. SECONDARY CATEGORY: n/a.