We reported that corticosterone (CORT) induces growth hormone (GH) cell differentiation in chicken embryonic pituitary cells in culture and in living embryos. The present study tested whether CORT could also induce prolactin (PRL) cell differentiation in vitro. CORT increased the percentage of GH cells in cultures of embryonic day 13 (e13) pituitary cells after 1 day of treatment and PRL cells after 3 days of incubation. Dual immunofluorescence showed that the PRL cells induced by CORT did not contain GH, indicating they were separate cell populations. Similar PRL cell responses were also observed in cultures of e11, e15 and e17 pituitary cells. ZK98299, a glucocorticoid receptor (GR) antagonist, suppressed CORT effects, verifying involvement of GR. Northern blot analysis indicated that CORT increased GH mRNA levels after one day of treatment. In contrast, increases in PRL mRNA levels were delayed and observed after three days of treatment. Induction of a luciferase reporter driven by the PRL promoter was also delayed until 3 days of CORT treatment. Dual labeling immunofluorescence indicated that the majority of PRL cells induced by CORT were not labeled with BrdU, suggesting that lactotrophs induced by CORT do not result from cell proliferation. Proportions of Pit-1 containing cells and the total amount of Pit-1 protein spontaneously increased with increasing culture time. However, no effect of CORT on Pit-1 levels or the number of Pit-1 containing cells were observed. We conclude that CORT can induce lactotroph differentiation in culture and that longer CORT exposure is needed for lactotroph induction compared with somatotroph induction. The effects of CORT on PRL cell induction are GR dependent and involve an increase in PRL gene expression. PRL cell induction by CORT is not associated with an increase in Pit-1 containing cells.