Serum prolactin was measured in 55 cows 2–4 hr before, immediately after and 1 hr after milking for the duration of lactation or week 44, whichever occurred first. Serum prolactin increased in response to stimuli associated with milking, but this response was largest at 8 weeks; of lactation (77 ng/ml) and then gradually decreased as lactation advanced until at 32 weeks prolactin was no longer released. As prolactin concentrations increased in the pre—milking serum samples the amount subsequently released by the milking stimulus was reduced (p < 0.01). The correlation between serum prolactin measured 2 to 4 hr before milking and milk yield was very low (r = -0.03), although similar calculations for samples collected immediately after and 1 hour after milking (r = 0.36 and 0.18, respectively) were significant (p < 0.01). Within—stageof— lactation correlations between serum prolactin and milk yield were very low and sometimes negative during early lactation, but after 12 to 24 weeks of lactation the correlations between the two variables were consistently positive and ranged from 0.08 to 0.48. Throughout pregnancy in 28 non—lactating heifers no consistent relationships were observed between serum prolactin and milk production during their subsequent lactation. Furthermore, stage of pregnancy during concurrent lactation of the 55 cows did not affect serum prolactin (p > 0.05) although lactating, non—pregnant cows had higher (p < 0.01) serum prolactin then lactating, pregnant cows. Neither sex of the fetus nor stage of the estrous cycle significantly affected serum prolactin in lactating cows. Serum prolactin was higher during April to September (74 ng/ml) than during October to March (35 ng/ml). However, prolactin released in response to milking was lower (19 ng/ml) in the summer than in the winter months (33 ng/ ml) and paralleled changes in milk yield. Similarly, multiparous cows produced more milk (p < 0.01) and had greater concentrations of serum prolactin (p < 0.05) than primiparous cows. We conclude that serum concentrations of prolactin were associated with changes in milk yield in the bovine, but cause—effect relationships remain to be established. (Endocrinology92: 1480, 1973)