The purpose of this study was to learn whether enriched populations of corticotropes could be grown without the other pituitary cell types. Corticotropes populations were enriched to 80-90% by counterflow centrifugation in an elutriator with the Sanderson chamber. After initial separation into small, medium, and large fractions, the cells were stimulated for 3 h with 0.5 nM corticotropin-releasing hormone (CRH) and then re-eluted to remove the enlarged corticotropes. More ACTH (6- to 10-fold) was released in media with 10% fetal bovine serum (FBS) than was released in media with no serum. The effects of FBS could not be mimicked by 0.3% BSA. Corticotropes grew in serum-containing media as long as they were plated at a density of at least 2500 cells per well. The corticotropes expanded in size and assumed two major morphological subtypes. Both stored ACTH and beta-endorphin. One subtype was flattened and pleomorphic. The other subtype was stellate with multiple processes. Cell counts showed a 2.5- to 3.8-fold increase in the number of labeled corticotropes during the first 21 days of culture. Then the numbers of cells declined rapidly. Basal secretion of ACTH rose 1.6-fold during the first week, plateaued after 14 days and then declined to less than 30% of first week levels. CRH stimulation produced dose-dependent increases in media ACTH. In 7 day cultures, both basal and stimulated levels of ACTH were similar to those in 7 day cultures of mixed pituitary cells (containing equivalent numbers of corticotropes). Stimulatory effects of CRH were evident for up to 42 days of culture. Arginine vasopressin enhanced CRH-mediated secretion in most cultures in the first week. Pretreatment with glucocorticoids (100 nM corticosterone) for 15 h blocked CRH-mediated secretion in all cultures. The studies showed that corticotropes do not need the other pituitary cell types for basic plating and basal and CRH-mediated secretory responses. Further tests of specific growth factors are needed to learn whether they will maintain function for longer periods.