To study the effect of müllerian inhibiting substance on testicular germ cell development, especially on gonocytes, whole testes (156) from newborn mice were cultured for 1 to 7 days in vitro. The synthetic medium contained either 10% fetal calf serum, which itself contains endogenous müllerian inhibiting substance, or transferrin, insulin and retinoic acid. Human recombinant müllerian inhibiting substance, rabbit antiserum against müllerian inhibiting substance and/or normal rabbit serum was added to some cultures. The cultured testes were fixed in Stieve's fixative and stained with hematoxylin and eosin, and the numbers and types of germ cells per tubule were counted under a light microscope. Preliminary studies showed that germ cell development in newborn mouse testes was similar in vitro to that observed in vivo, except for delay in vitro. Normal germ cell maturation from gonocytes to primary spermatocytes occurred in testes cultured with 10% fetal calf serum only (i), 10% fetal calf serum plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (ii), 10% fetal calf serum plus normal rabbit serum (iii) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance (iv). Maturation from gonocytes to A-type spermatogonia was arrested in testes cultured with 10% fetal calf serum plus anti-müllerian inhibiting substance antibody (p < 0.01), transferrin, insulin and retinoic acid alone (p < 0.001) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (p < 0.001). The results are consistent with the hypothesis that müllerian inhibiting substance may be involved in postnatal gonocyte development and suggest that it may be useful to treat infertility associated with undescended testes.