The IGF-II gene is a complex transcription unit. Multiple transcripts are synthesized as a result of alternate promoter usage and the splicing of unique 5' untranslated regions to common coding exons. In order to characterize the mechanisms of IGF-II gene regulation we performed comparative studies to define essential features of IGF-II expression in human, rat and mouse. Homologous promoter regions of the human, mouse and rat IGF-II genes were fused to the luciferase reporter gene and expression was measured in various cell lines that have an endogenously active or inactive IGF-II gene expression pattern, respectively. The transient promoter activity of the human, mouse and rat IGF-II constructs was further compared with the endogenous activity of the IGF-II gene in various tissues and cell lines of human, mouse and rat origin. The results indicate that in transient expression assays employing heterologous systems (e.g., mouse promoter in human cells), most IGF-II promoter constructs are active, albeit at low levels. Maximal promoter activity is only observed, however, in homologous systems (e.g., human promoter constructs tested in human cells). This suggests that each promoter, despite the strong sequence conservation of the homologous human, rat and mouse promoters, is adapted to the levels of the transcription factors present in its natural environment. Finally, IGF-II gene expression is not only regulated at the level of transcription but also depends on mRNA stability. We show that human, rat and also mouse IGF-II mRNAs are subjected to specific endonucleolytic cleavage, suggesting that specific cleavage of IGF-II mRNAs must be of general physiological importance.