Gonadotropes from cycling female rats were studied to investigate possible mechanisms for the nonparallel release of LH and FSH. The percentages of total gonadotropes increased from 14% during estrus (E) to 18% by diestrous day 2. More of these cells became multihormonal on the morning of proestrus (P; from 46% during diestrus to 69%). Since LH-containing cells increased from 7% at E to 13.3% during early proestrus, this suggests that monohormonal FSH cells may have contributed by synthesizing LH. Gonadotrope cell areas were greatest just before the LH surge (P 1600 h). Microdensitometric measurements demonstrated that the amount and density of immunoperoxidase stain for either gonadotropin subunit were highest during the midafternoon of P. Interestingly, the amount of stain for LH continued to increase during the LH surge, suggesting that the stain had detected newly synthesized LH beta. At the same time, the average density of the LH beta stain decreased. In contrast, the amount, concentration, and density of stain for FSH beta increased during the afternoon of P and decreased during late P and early E. The percentages of granules that contained immunogold stains for only LH or FSH (monohormonal granules) at P 1600-P 1700 h were 3-4 times higher than those in diestrous rats. The percentages of monohormonal LH granules declined during the proestrous surge, whereas percentages of monohormonal FSH granules declined during the first rise (P 1900 h) and after the second rise in serum FSH (E 0800 h). Finally, the average number of gold particles per micron 2 granule area rose over the value in diestrous rats during P 1600-P 1700 h. These studies suggest that multihormonal gonadotropes support nonparallel gonadotropin release by changing the rate of subunit packaging and transit in the Golgi complex.