Bovine anterior pituitaries were dispersed with collagenase and the cells grown to monolayers. On day 3 or 4 or culture, cells (4 flasks/treatment group) were incubated for 2 hr with TC medium 199, then with TC medium 199 containing thyrotropin releasing hormone (TRH) for 2 hr. Relative to to values during the first 2-hr incubation, prolactin concentration in media from cow pituitary cell cultures exposed to 0 (control), 0.01, 0.1 or 10.0 ng TRH/ml medium average -23, 799, 1966, 1926 and 1976 ng/ml. Relative to control, each dose of TRH increased (P less than 0.01) media prolactin concentration. Comparable values for growth hormones were -5, 9, 12, 21, and 21 ng/ml and the increase in growth hormone release relative to controls was significant (P less than 0.05). A second experiment, designed to determine whether TRH would increase prolactin release by pituitary cells from cows, steers and a bull was conducted using the same procedures. Changes in media prolactin concentration (ng/ml) after 0, 0.01, 0.1, 1.0, 10.0 and 100.0 ng TRH/ml medium was -27, 63, 109, 207, 226 and 137 for cows and -14, 13, 36, 153, 166 and 80 for steers. After 0.0, 10 and 100 ng TRH/ml medium comparable values for a bull were -6, 14 and 31 ng/ml. Growth hormone (ng/ml) in these media was not different from controls. We conclude that TRH stimulates prolactin release from pituitary cells of cows, steers and bulls but its effect on growth hormone release is not consistent.