Sertoli cells dissociated from 10-day-old rat testes form colonies in primary culture in response to FSH. FSH and dibutyryl cAMP stimulated the attachment of Sertoli cells to an equal extent; however, FSH-treated cultures contained a small number of large colonies while dibutyryl cAMP-treated cultures contained a large number of small colonies. This relationship was not altered by the addition of a number of other peptide or steroid hormones. Extracellular cAMP levels and colony density were negatively correlated. Colony size at 24 h of culture was diminished in FSH-treated cultures by the addition of a cAMP antibody at 6 or 12 h of incubation. The addition of cAMP at 24 h to FSH-treated cultures caused a dose-dependent stimulation of colony size but not colony density at 48 h of culture. A point source of cAMP (4 x 10(-3) M in agar) inhibited migration of cells toward the agar spot. An agar spot on the dish substratum containing Sepharose-bound FSH exhibited a halo of cells next to the spot, with a zone lacking cells distal to it. Radioautographs of [125I]iodo-FSH-treated cultures exhibited a nonhomogeneous distribution of silver grains; colony size increased faster than the number of labeled cells. Taken together, the results suggest that 1) FSH is stimulating the attachment of Sertoli cells through an increase in intracellular cAMP, 2) FSH is promoting active aggregation of Sertoli cells in culture through a modulation of extracellular cAMP, and 3) cells with a large amount of bound FSH are acting as centers for aggregation.