The existence of differences in the amount of hormone released by individual lactotropes from the same pituitary gland has been unequivocally established. Such secretory heterogeneity could reflect the existence of lactotrope subtypes that differ quantitatively in their capacity to release constant amounts of hormone over time. Alternatively, the same pattern could arise from the intermittent secretion of hormone by individual cells. In an attempt to distinguish between these possibilities, we used a novel version of a sequential plaque assay to monitor daily the amount of PRL secreted by the same individual cells over a period of 4 days. When more than 1200 cells from male and lactating female rats were analyzed in this manner, we found that the average amount of hormone released over time was relatively constant within gender and that (as anticipated) cells from females released 2.6-fold more PRL than did those from males. However, assessment of secretory variability by individual lactotropes revealed that male cells were considerably more irregular in their pattern of PRL release than were their female counterparts. Indeed, only 11.8 +/- 2.5% (mean +/- SEM) of male cells secreted relatively constant amounts of hormone over the 4-day period, whereas 43.4 +/- 8.4% of the female cells exhibited a similar profile (assessed by comparing coefficients of variation for individual cells). Interestingly, this variability in secretion seemed to be inversely related to the amount of hormone released; constant secretors had a much higher average plaque size (2.5-fold greater; P < 0.01) than irregular secretors. Taken together, our results demonstrate that individual lactotropes from the same animal can release hormone in either a constant or episodic manner, and that the gender of the pituitary donor can have a strong influence on the relative abundance of each phenotype. Moreover, our data suggest the former phenotype makes a greater contribution to overall PRL secretion than the latter.