Factors responsible for development of catecholamine (CA) responsiveness in granulosa cells (Gc) of preovulatory follicles from immature rats injected with 10 IU of pregnant mare's serum gonadotropin (PMSG) on Day 26 were studied. CA did not stimulate cyclic AMP (cAMP) production in whole follicles isolated before (morning) or after (evening) the preovulatory gonadotropin surge on Day 28, while newly formed corpora lutea found on Day 29 responded to CA. Gc from the preovulatory follicles did not respond to CA when tested immediately after isolation. Gc were cultured for various periods in Eagle's MEM without serum and subsequently tested for a possible stimulation of cAMP and steroidogenic responses by CA. In Gc from follicles isolated in the morning (AM-Gc) and cultured for 12 or 24 h, no response to CA was found, while the Gc isolated in the evening (PM-Gc) and cultured similarly for 12 h showed a marked response to CA both in stimulation of cAMP and progesterone production. By a total or partial elimination of the gonadotropin surge, using pentobarbital in combination with luteinizing hormone (LH) or using specific antisera to LH or follicle-stimulating hormone (FSH), it was found that previous exposure to LH was necessary for the PM-Gc to develop CA responsiveness during culture. Further, it was possible to induce CA responsiveness in AM-Gc by treatment of rats with LH for a short period in vivo followed by a period of culture. The appearance of CA responsiveness in PM-Gc cultured for 12 h was abolished when cycloheximide (5 micrograms/ml) was present during culture. It appears that the following three conditions have to be satisfied for the isolated Gc to develop CA responsiveness: 1) exposure to LH in vivo, 2) culture for a short period, and 3) an active protein synthesis. It is concluded that under physiological conditions the process of luteinization is associated with acquisition of responsiveness to CA and this process depends on the LH component of the preovulatory gonadotropin surge.