Small and medium-sized gonadotropes may enlarge and produce more LH in order to contribute to the proestrous surge. To test this hypothesis, dispersed pituitary cells from cycling female rats were separated by centrifugal elutriation into small, medium, and large fractions and labeled for LH beta antigens or mRNA (by in situ hybridization with a biotinylated oligonucleotide probe complementary to sequences encoding amino acids 28-40). The percentage of cells bearing LH beta mRNA in the pituitary cell population increased from 6 +/- 0.4% in the evening of diestrous day 2 to 16 +/- 0.7% in the morning of estrus (average +/- SEM). Over 80% of these labeled cells were large or small subtypes. The proportion of small gonadotropes labeled with LH beta mRNA declined from 43 +/- 3% at metestrus to 29 +/- 1% on the evening of proestrus as the proportion of medium-sized gonadotropes labeled for LH beta antigens (15 +/- 1%) or mRNA (17 +/- 1%) increased to 25 +/- 2% or 38 +/- 2%, respectively. Because the overall percentage of immunoreactive LH cells did not change after diestrus, small LH cells may have enlarged or increased their density to join the medium-sized pool. During proestrus, the proportion of large immunoreactive LH gonadotropes increased from 41 +/- 2% to 65 +/- 2% (by the morning of estrus) as the proportion of small or medium-sized LH cells declined to 17-18 +/- 1%, suggesting further increases in size or density. These data suggest that small or medium-sized gonadotropes are activated during early diestrus to enlarge and produce LH beta. They contribute to the increased number of cells in medium-sized and large fractions in proestrous or estrous rats. The predominance of the smaller subtypes during metestrus and diestrus suggests that LH gonadotropes may revert to a smaller or lighter subset to await activation during the next cycle.