The stimulatory action of estrogens on prolactin (PRL) secretion and synthesis is well known; on the other hand, anti-calmodulin drugs have recently been shown to inhibit prolactin in vitro release induced by estrogens. Based on these data, we decided to evaluate the in vivo effect of anti-calmodulin drugs (trifluoperazine and W7) on basal and estradiol-17 beta stimulated levels of PRL mRNA in anterior pituitary lobes obtained from adult male rats. Total RNA was isolated from pooled pituitaries recovered from animals under the same treatment and, from it, hybridizable PRL mRNA was detected. Estradiol-17 beta consistently stimulated PRL mRNA levels by 3-4 fold. The utilization of either trifluoperazine or W7, invariably inhibited estradiol-17 beta stimulated PRL mRNA. Metoclopramide, a drug with antidopaminergic activity, potentiated the stimulatory effect of estradiol-17 beta on PRL mRNA levels. These results suggest that anti-calmodulin drugs have an in vivo antiestrogenic effect on PRL mRNA levels confirming previous in vitro studies. Although, it is difficult to be conclusive about the mechanism through which these drugs act, one possibility is that the calcium-calmodulin system may be involved.