The obligatory role of follicle stimulating hormone (FSH) in normal development and function of antral follicles in the mammalian ovary is well recognized but its function in preantral growth is less clear. The specific objective of this study was to investigate the response, in culture, to FSH of mouse preantral follicles of increasing size, focusing particularly on growth rate and gene expression. Preantral follicles were mechanically isolated from ovaries of C57BL/6 mice, 12-16 days post partum and single follicles cultured for up to 96h in medium alone (n=511) or with rhFSH 10ng/ml (n=546). Data were grouped according to initial follicle diameter in 6 strata ranging from <100 to >140 μ m. Follicles of all sizes grew in the absence of FSH (p<0.01, paired t-test). All follicles grew at a faster rate (p<0.0001) in the presence of 10 ng/ml FSH but larger follicles showed the greatest change in response to FSH. Even the smallest follicles expressed FSH receptor mRNA. FSH-induced growth was inhibited by KT5720, an inhibitor of protein kinase A (PKA), implicating the PKA pathway in FSH-induced follicle growth. In response to FSH in vitro, FSH receptor mRNA (measured by qPCR) was reduced (p<0.01) as was Amh (p<0.01) whereas expression of StAR (p<0.0001) and the steroidogenic enzymes Cyp11a1 (p<0.01) and Cyp19 (p<0.0001) was increased. These results show heterogeneous responses to FSH according to initial follicle size, smaller follicles being less FSH-dependent than larger preantral follicles. These findings strongly suggest that FSH has a physiological role in preantral follicle growth and function.