Anterior pituitary corticotropes represent only 9-10% of the mixed pituitary cell population. However, their small size precludes their enrichment because they cannot be separated from the more abundant PRL and GH cells. They can be induced to enlarge by adrenalectomy, and this report describes the separation of larger CRH-responsive corticotropes from a subpopulation of small pituitary cells. The separation was done by counterflow centrifugation in an elutriator containing the Sanderson chamber which was designed to separate small cells under 15 micron in diameter. The corticotropes were initially eluted at flow rates under 30 ml/min along with other cells less than 12.5 micron in diameter. They were then stimulated for 2-4 h with 0.5 nM CRH and reeluted with the use of higher flow rates to separate the enlarged corticotropes from the unstimulated cells. Reelutriation of the entire pool of small cells produced an enrichment to 60% corticotropes in five separate experiments. However, when the pool was divided into smaller cells (eluted at 20 ml/min) and medium-sized cells (eluted at 30 ml/min), and the two pools were reeluted separately, the enrichment increased to over 90% corticotropes in eight separate experiments. These corticotrope populations remained enriched for up to 14 days in culture. They also secreted in a reverse hemolytic plaque assay that recognizes ACTH-(25-39). The dual labels for ACTH and beta-endorphin showed that 60% of the corticotropes stored both peptides, whereas 30% stored only ACTH, and 10% stored only beta-endorphin. No differences in storage patterns were seen when small and medium-sized corticotropes were compared. Thus, these studies present the first report of the production of an enriched fraction of CRH-responsive corticotropes by counterflow centrifugation and the first report of heterogeneous storage of ACTH and beta-endorphin. The use of enriched fractions facilitated the analysis of these heterogeneous storage patterns in over 8000 corticotropes.