Expression of the cloned rat growth hormone gene was studied in transfected CV-1 cells. Cell lines expressing rat growth hormone synthesized and secreted the 22 kilodalton form and the 20 kilodalton variant. The results confirm that the 20 kilodalton variant arises from an alternatively spliced mRNA rather than expression from a variant gene in rat. The 5' end of the mRNA from expressing cell lines was located upstream of the normal initiation site in rat. Transcription initiated within a 5' flanking AT rich sequence. Results presented indicate that transcription from normally silent promoter or promoter-like sequences dictated rat growth hormone gene expression in transfected cells. Finally, no hormone was expressed by CV-1 cells transfected with a plasmid containing the rat growth hormone gene in the same transcriptional reading frame as the neomycin resistance gene of pSV2neo indicating an effect of cloning orientation on expression in CV-1 cells.