Recent evidence indicates that the human and rodent beta 3-receptor genes differ with respect to their intron/exon organization and putative regulatory elements in the proximal promotor regions. The present work examined the molecular basis for heterogeneity among human and rodent beta 3-receptor messenger RNAs (mRNAs) and mRNA transcription start site selection with gene-derived human and rodent probes. This analysis indicates that the mRNA size heterogeneity of the human beta 3-receptor mRNA seen in Northern blots results from the use of alternative polyadenylation signals. In contrast, mRNA heterogeneity of the rat beta 3-receptor mRNA is due to the use of widely separated promoters. Analysis of mRNA transcription sites within the proximal promoters of the human and rat genes indicates that multiple homologous start sites are used. However, the pattern of transcription initiation appears to be both species and tissue specific. In rat adipose tissues where abundant expression of the beta 3 gene occurs, most transcripts began at -161 relative to translation initiation ATG, whereas minor transcription began around -123 and -109. Interestingly, virtually all transcripts were found to begin at the -123 and -109 sites in the rat gastric fundus, which expresses only low levels of beta 3-receptor mRNA. In human brown fat and neuroepithelioma cells, the vast majority (> 80%) of beta 3 transcripts began at multiple sites around -130. Exposure of murine 3T3-F442A adipocytes to isoproterenol, 8-bromo-cAMP, phorbol 12-myristate 13-acetate, or dexamethasone dramatically reduced murine beta 3-receptor mRNA levels. In contrast to mouse adipocytes, 8-bromo-cAMP increased human beta 3-receptor mRNA levels in SK-N-MC neuroepithelioma cells, whereas isoproterenol, phorbol 12-myristate 13-acetate, and dexamethasone were without effect. These data identify important differences in the structure and regulation of the human and rodent beta 3-receptor genes and suggest that the data obtained in rodent models may not be directly applicable to the regulation of the human gene.