A biotin-conjugated synthetic corticotropin releasing factor (B-CRF) was prepared and characterized. Its biological activity and binding affinity were compared with that of unlabeled synthetic CRF. Both forms of the releasing factor were equipotent in in vitro studies measuring the release of corticotropin (ACTH) (ED50 = 1 nM). The IC50 in the binding assays was 1.5 nM for CRF and 4 nM for B-CRF. Dual avidin-biotin peroxidase complex stains were then used in pituitary monolayer cultures to visualize receptivity to the releasing factor and to confirm opiocortin storage in the target cells. All corticotropes showed stain for B-CRF. The percentage of cells that were double-labeled for ACTH and CRF increased with the dose of B-CRF during a four hour incubation period. The CRF stain was abolished, however, when an excess of unlabeled CRF was added to compete with B-CRF. The distribution of the B-CRF and ACTH stains varied in the cells with the time of exposure to the analog. These studies show that biotin-conjugate CRF is a potent analog that can be demonstrated cytochemically on cells identified immunocytochemically as corticotropes. It can be used to follow important events associated with CRF stimulation including the rapid internalization of CRF coupled with the mobilization of corticotropin stores and the formation of cellular processes.