Structural analysis of 55 nearly full-length cDNA clones revealed heterogeneity in the 5'-untranslated regions of rat liver alkaline phosphatase mRNAs. The 5' extremities diverged into two totally unrelated sequence stretches at the position 88 nucleotides upstream of the initiation codon ATG. These two sequences, referred to as E1 and E2, were assigned on the genome about 36,000 base pairs (36 kbp) and 10 kbp upstream, respectively, of the exon coding for the 5'-most part of the common region. The gene consisted of 13 exons, including E1 and E2, and spanned about 56 kbp. The 11 exons (E3 to E13) following E1 and E2 were shared in common by the E1-type and the E2-type mRNAs. Analyses by S1 nuclease mapping and primer extension revealed the presence of two independent transcription-initiation sites specific to each of the E1 and E2 sequences. These results are interpreted as indicating a possible alternative usage of two leader exons, hence the presence of two independent promoters. Structural features of these putative promoters are described in the context of transcriptional fundamental and regulatory cis-elements.