Alternative splicing of the rat type 1 pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PVR1) produces variants that couple either to both adenylyl cyclase (AC) and phospholipase C (PLC) (PVR1 short, PVR1 hop, PVR1 hiphop), or to AC alone (PVR1 hip). We have previously shown that populations of clonal T3-1 gonadotrophs express PVR1 hop and PVR1 short mRNAs, whereas clonal GH 4 C 1 somatotrophs do not. Here we have used the single cell RT-PCR technique to investigate whether normal rat gonadotrophs and somatotrophs express PVR1 mRNA, whether a single cell co-expresses multiple splice variant forms, and whether differential PVR1 mRNA expression correlates with differences in PACAP-stimulated Ca 2+ signalling. We found that individual rat gonadotrophs expressed mRNA either for PVR1 hop, for PVR1 short, or co-expressed the two forms. Although we found no differences between the splice variant(s) expressed and the characteristics of PACAP-stimulated Ca 2+ responses, the expression of PVR1 mRNA is consistent with the known PACAP stimulation of the PLC system in gonadotrophs. Individual rat somatotrophs also expressed PVR1 hop or PVR1 short (but not PVR1 hip) mRNAs although these forms were never co-expressed. The expression of PVR1 mRNA in somatotrophs can explain in part the activation by PACAP of the AC system in such cells. In conclusion, the single cell RT-PCR technique was used to demonstrate expression of multiple PVR1 splice variants in single identified pituitary cells. These findings open up important questions on the role of alternative splicing in cell biology.