Arginine vasopressin (AVP) stimulates the secretion of ACTH and TSH. Recently, affinity cytochemical techniques with a potent biotinylated analog of AVP were used to identify ACTH cells as one of the target cells. Counts showed that AVP-bound cells were 10% of the population. However, if AVP bound all corticotropes and thyrotropes, one would expect AVP to bind at least 16% of the pituitary cells. Therefore, dual cytochemical labeling protocols were used to learn if thyrotropes also bound AVP (bio-AVP). Forty-eight percent of AVP target cells contained ACTH, and 42% contained TSH beta. The percentages of AVP-bound cells were increased to 12-13% of the total pituitary cells after 1-h pretreatment in 10 nM TRH or CRH. TRH and CRH together stimulated increases to 16% of the total cells. Analysis of dual labels showed that the additional AVP-bound cells stimulated by CRH or TRH stemmed from the corticotrope or thyrotrope populations, respectively. TRH stimulated an increase in the percentage of TSH cells that bound AVP from 55% to 75%. Similarly, CRH stimulated an increase in the percentage of ACTH cells that bound AVP from 61% to 79%. In addition, the populations of cells labeled for TSH beta or ACTH antigens increased by 30% after 1 h in unlabeled AVP, supporting its direct effect on these target cells. TRH stimulated a similar increment in TSH cells. CRH pretreatment had no effect on the percentages of cells labeled for TSH or ACTH. This could be the result of loss of ACTH stores needed to identify stimulated corticotropes. Finally, analysis of the total percentages of AVP-bound TSH beta or ACTH cells suggested an overlap in the population. This stimulated the application of dual labels for ACTH and TSH beta. In populations exposed to vehicle only, 1-2% of mixed pituitary cells stored both ACTH and TSH. This unique cell type also comprised 10% of a corticotrope population enriched by counterflow centrifugation. The percentage of ACTH-TSH cells in the mixed cell population was augmented to 4.8% after 1 h in AVP. It was not affected by exposure to either TRH or CRH (or both peptides). These studies demonstrate that AVP target cells include thyrotropes, corticotropes, and unique cells that store both ACTH and TSH.