The effect of transferrin on basal and FSH-stimulated aromatase activity of granulosa cells from immature female rats treated with diethylstilboestrol (DES) was examined in vitro by a radiometric method. The basal activity of the enzyme was very low after 3 days of incubation. Treatment with FSH (20 ng/ml) resulted in a 9.6-fold increase in activity, whereas coincubation with increasing doses of transferrin (3-300 micrograms/ml) produced a dose-dependent inhibition of FSH-stimulated aromatase activity with a projected minimal effective dose of less than 2 micrograms/ml. A time-course study showed that the inhibitory effect of transferrin on aromatase activity has become significant at 48 h of incubation. The inhibitory action of transferrin on the enzyme complex was further confirmed by showing that the FSH dose-response curve was significantly suppressed by concomitant treatment with 100 micrograms transferrin/ml with a maximum suppression of 54.1% at a dose of 30 ng FSH/ml. The possibility that transferrin may act through a non-specific inhibitory effect seems unlikely, as no changes in cell number and DNA content per well were observed. In fact, protein synthesis was enhanced after treatment with transferrin. Aromatase activity, stimulated by several promoters of cyclic AMP (cAMP), such as prostaglandin E2 (PGE2), forskolin and 8-bromo-cAMP, was significantly suppressed by 100 micrograms transferrin/ml (36.6, 47.4 and 23.4% inhibition respectively), suggesting that the effect of transferrin on FSH action may involve a site(s) distal to cAMP generation. These findings indicated that transferrin, present in follicular fluid, may play an important role in the regulation of granulosa cell differentiation.