The contribution of dietary, mobilized, or newly synthesized fatty acids in yolk formation at different periods of egg production was determined. In an initial experiment, a single dose of 13C-linoleic acid was administered to pullets at the onset of egg production and their presence in follicles determined over the subsequent 10 days. In a second experiment, pullets were fed a daily 15 mg dose of U-13C-glucose beginning 2 wk prior to sexual maturity through the end of the experimental period. A 50 mg meal of U-13C-linoleic acid was orally administered approximately 10 d prior to sexual maturity (defined as first egg) representing body linoleic acid. Upon each hen's first egg, each bird received a 25 mg meal of 2D31-linoleic acid representing dietary linoleic acid. All eggs were collected for the next 10 days. The incorporation of labeled linoleic acid and palmitic acid in egg yolk was then determined using GC-MS. This process was repeated at peak production and at 45 wk of age. At sexual maturity, the deposition of labeled palmitic acid in the yolk was higher compared with its deposition at peak production and 45 wk of age. The deposition of both 13C- and 2D31-linoleic acid increased with hen age. These results suggest that dietary and tissue linoleic acid is utilized to a greater extent in older hens and that lipogenesis (synthesis of palmitic acid) plays a larger role at sexual maturity in the young hen.