LH secretion from pituitary cell fractions separated by centrifugal elutriation was compared to learn whether any contributed to the LH surge. After plating (1 h) and stimulation with 0-10 nM [D-Lys6]GnRH (4 h), some fractions secreted levels that were out of proportion to their enrichment. Large cells from proestrous morning rats (3-fold enriched) secreted 4.3 times more LH, and medium-sized fractions (1.5-fold enriched) secreted 2-3.4 times more LH than unseparated cells during estrus or metestrus. Normalized data (nanograms per 20,000 cells) showed that basal levels reflected the enrichment in the fractions. Data were also normalized to nanograms of LH per 1,000 LH gonadotropes to focus on LH cell activity. Basal secretion in unseparated cultures (4-6 ng/ml/1,000 LH cells) was lower than that in small or large LH cells during all stages except proestrus, when small gonadotropes became as active as those in unseparated cultures, and large gonadotropes secreted 2-3 times more LH. Basal secretion from medium-sized gonadotropes was comparable to that in unseparated cultures. [D-Lys6]GnRH-mediated secretion from unseparated, small, and large LH cells was comparable during most stages. However, during proestrus, large gonadotropes secreted 2.2 times more LH than unseparated counterparts. Stimulated medium-sized LH cells were 1.3-2.3 times more active in most stages than those in unseparated cultures and 1.75-2.8 times more active than those in large cell fractions (from proestrous PM to metestrus). Whereas this enhanced secretion late in proestrus suggests that medium-sized LH cells may support the LH surge, it also may reflect the removal of regulatory factors from cells in other fractions. Studies of autocrine or paracrine interactions with gonadotropes are needed to test this hypothesis.