To study the control of prolactin secretion in fish, an in-vitro technique using a monolayer cell culture system of rainbow trout pituitary glands was developed. Such secretion was characterized by measurement of both prolactin release and prolactin mRNA content using a trout prolactin cDNA as a probe. This cell culture technique, already used to study the regulation of gonadotrophin secretion in rainbow trout, was further validated by measuring total DNA and protein content. Both parameters appeared to be stable after 2 days of culture. Studying the effect of somatostatin (SRIF) on prolactin cells indicated that a maximal inhibitory effect (62%) was observed after 24 h of treatment. Significant inhibition of prolactin release was obtained for SRIF doses ranging from 50 nm to 1 μm. However, in the same experiment, SRIF was much more potent as an inhibitor of growth hormone release. Short-term (<12h) incubation with SRIF did not induce a significant change in prolactin release, whereas growth hormone release was reduced at as early as 1 h after SRIF exposure. SRIF did not have a significant effect on total prolactin content or prolactin mRNA levels, suggesting the absence of an effect on prolactin synthesis. No increase in the magnitude of the inhibitory effect of SRIF was observed when using pituitary cells from immature, mature male or mature female trout. When comparing effects on primary cultures containing cells from the whole pituitary with a prolactin cell-enriched population, SRIF appeared to have the same inhibitory effect on prolactin release, supporting a direct action of SRIF on prolactin cells. These results provide further support for SRIF being a prolactin-inhibiting factor in rainbow trout and acting as a modulator of a dominant stimulatory control of prolactin release.