Serum prolactin (Prl) was measured following decapitation at various stages of the estrous cycle in control animals and following cervicovaginal stimulation with a glass rod. Serum Prl was low at 10.00 h at all stages of the estrous cycle but was elevated on the afternoon of proestrus with peak values at 16.00 h and on the afternoon of estrus with peak values at 13.00 h. Cervicovaginal stimulation resulted in 100% incidence of pseudopregnancy when performed on estrus and diestrus I and in a still high incidence when performed on proestrus. However, on diestrous day II, it caused only delayed pseudopregnancy in less than half the rats. Serum Prl was significantly elevated in the afternoon following stimulation on diestrous day I and on estrus, but the already elevated values on proestrus were not further augmented and there was no significant elevation on diestrous day II. Thus, there was a cyclic variation in the response of Prl to cervicovaginal stimulation which correlated with the incidence of immediate pseudopregnancy. Gonadal steroids at this stage of the cycle probably modulate the reflex release or Prl either by an action at the hypothalamic or pituitary level or at both sites. On the basis of evidence provided by others, the immediate release of Prl does not appear to be required for the induction of pseudopregnancy.