Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor which has been proposed to exert its secreting property by activating the adenylate cyclase enzyme. The present study shows that the omission of external Ca<sup>2+</sup> did not affect the ability of VIP to induce PRL release while it completely abolished the VIP stimulatory effect on adenylate cyclase. We found that VIP (500 nM) stimulated PRL secretion in a time-dependent manner reaching a plateau at 3 min. This pattern was not changed when Ca<sup>2+</sup> was omitted from the incubation medium. When tested at different concentrations, VIP stimulated PRL release with EC<sub>50</sub> values of 1.3 n M in the presence of Ca<sup>2+</sup> and 30 n M in the absence of Ca<sup>2+</sup>. On the other hand, Ca<sup>2+</sup> removal completely suppressed the VIP-induced cAMP formation. VIP (200 n M) was also found to activate Ca<sup>2+</sup> influx into pituitary cells. The increase in Ca<sup>2+</sup> permeability showed a peak at 5 s and remained significantly higher than control values until 1 min. In conclusion, in an experimental condition where Ca<sup>2+</sup> was omitted from the medium, VIP was found to induce PRL release without stimulating cAMP production. This cAMP-independent PRL release was blocked by preincubation of the cells with 1 µg/ml pertussis toxin. An additional mechanism other than adenylate cyclase activation or Ca<sup>2+</sup> entry is proposed to sustain VIP-induced PRL release.