FSH regulation of inhibin α-, β_B-subunit and follistatin mRNA was investigated in cultured chicken granulosa cells, which were isolated and pooled according to size from the F₄ F₅ follicles, small yellow follicles (SYF), and large white follicles (LWF). In experiment 1 (four replicate experiments), granulosa cells were cultured, and the effect of FSH (50 ng/ml) on the growth of cells from the different follicles was examined at 24 and 48 h of culture. Cell viability was >95% for all of the granulosa cell cultures at 24 and 48 h. At 24 h, the number of granulosa cells in both the FSH-treated and the untreated cultures for all follicle types was numerically greater than the number of cells originally plated. At 48 h, FSH-treated cultures for all follicle types had twice (P < 0.05) the number of cells as the untreated cultures. In experiment 2 (three replicate experiments), FSH increased expression of the mRNA for inhibin α-subunit in LWF granulosa cells at 4 and 24 h to detectable levels and increased inhibin α-subunit protein accumulation to detectable levels by 24 h in granulosa cells from the LWF. FSH also increased (P < 0.05) mRNA levels for the inhibin α-subunit at 4 and 24 h in SYF granulosa cells and at 24 h in F₄ F₅ granulosa cells. The effects of FSH on follistatin and β_B-subunit were variable with respect to follicle development and culture duration. These results suggest that FSH plays an important role in stimulating the production of mRNA and protein for the inhibin α-subunit in small prehierarchical follicles.