Blood was collected, at 5-min intervals for 3 h, via jugular cannulation every 14 days during the first 4 months post partum from beef cows and heifers, 4 suckling 2 calves and 4 suckling 1 calf. Calves were isolated from the dams to prevent suckling for approximately 4 h before and 2 1/2 h during sampling to obtain baseline values for prolactin, measured by radioimmunoassay. During the last 30 min of sampling, calves were allowed to suck. Milk samples were also collected at 28-day intervals from 60 females, 30 with twins and 30 with single calves. beginning 2 weeks after calving and continuing until calves were weaned at 180 days of age. No differences were observed between dams with 1 and 2 calves for baseline plasma prolactin level or for prolactin response to the suckling stimulus by 1 or 2 calves. However, milk prolactin concentration was significantly higher (P less than 0.01) for dams with 2 calves, probably reflecting the more frequent suckling that occurs with twins. Milk prolactin value in this study was not highly correlated with the post-partum interval to first ovulation from another study on these animals. It is concluded that prolactin is not significantly higher (P less than 0.01) for dams with 2 calves, probably reflecting the more frequent suckling that occurs with twins. Milk prolactin value in this study was not highly correlated with the post-partum interval to first ovulation from another study on these animals. It is concluded that prolactin is not significantly higher (P less than 0.01) for dams with 2 calves, probably reflecting the more frequent suckling that occurs with twins. Milk prolactin value in this study was not highly correlated with the post-partum interval to first ovulation from another study on these animals. It is concluded that prolactin is not the primary factor controlling the longer post-partum interval to first ovulation in beef cattle with twins.