Here we describe an RT-PCR analysis of mono-ADP-ribosyltransferase 3 (ART3) mRNA expression in macrophages, testis, semen, tonsil, heart and skeletal muscle and the complete gene structure as obtained by sequence alignment of PCR products with a human genomic clone (GenBank accession no. AC112719). Twelve exons (ex1-12) were found to make up the coding region of the gene (one more than previously published). Two prominent classes of ART3 splice variants could be distinguished by the presence or absence of ex2 which encodes most of ART3 protein. Among the ex2-containing mRNA species, the most frequently amplified variant did not include exons 9 to 11, except in skeletal muscle, in which the major splice variant lacked ex10 only. Two different, previously not reported 5' non-translated regions (5' UTRs) were identified, demonstrating the presence of two alternative promoters that we termed palpha and pbeta. Whereas the 5'UTR originating from palpha, was split up into three exons, a single exon represented the 5' UTR of pbeta transcripts. Strikingly, in heart, skeletal muscle and tonsils the upstream promoter palpha was totally inactive and ART3 transcription appears to be driven solely by pbeta. In all other cell types tested, transcription started mainly (if not exclusively) at palpha. Thus, ART3 expression in human cells appears to be governed by a combination of differential splicing and tissue-preferential use of two alternative promoters. This specific use is evolutionary conserved as shown by analysis of the 5' UTR of the mouse ART3 mRNA.