The IM-9-P3 family of cell lines, which are derived from the B-lymphoblastoid IM-9 cell line, transcribe the human PRL (hPRL) gene by utilization of the decidual-type promoter and provide a model to study factors controlling extrapituitary expression of the hPRL gene. Here we describe regulation of hPRL gene expression in members of the IM-9-P3 family by retinoic acid (RA). When cells were incubated in medium supplemented with fetal calf serum that had been treated with dextran-coated charcoal, the addition of RA caused a 2-fold stimulation of hPRL secretion in the low hPRL-producing clone IM-9-P31 and the moderate producer IM-9-P32 (ED50, 0.53 and 0.13 nM, respectively), but not in the high hPRL-producing IM-9-P33 clone. Secretion from the RA-responsive cell lines increased steadily over the first 24 h of exposure and remained elevated for several days. The concomitant increase in hPRL mRNA steady state levels was not due to enhanced transcription of the hPRL gene, as assessed by nuclear run-on experiments, but, rather, to message stabilization. In RA-treated IM-9-P32 cells, the half-life of hPRL mRNA was significantly increased from 9 to 22 h. The transcripts were found to be preferentially associated with membrane-bound polysomes, thus being available for the secretory pathway. When we studied the expression of potential transducers of the RA signal, namely the RA receptor subtypes hRAR alpha, -beta, and -gamma and cellular RA-binding protein, we did not detect hRAR gamma or cellular RA-binding protein transcripts in the hPRL-negative clone IM-9-P6 or the hPRL-positive clones IM-9-P31, IM-9-P32, and IM-9-P33. hRAR alpha was equally expressed in all cell lines and not regulated by RA, whereas hRAR beta was differentially expressed and controlled by RA. This receptor subtype was absent from hPRL-negative members of the IM-9-P family, strongly induced by RA in the RA-responsive IM-9-P31 and IM-9-P32 cell lines via rapid transcriptional up-regulation, and only slightly induced in the RA-resistant IM-9-P33 cell line, suggesting a function in mediation of the effect of RA on hPRL gene expression.