We undertook cloning and sequencing of the 5' portion of the human aldolase A gene to elucidate the mechanisms that govern synthesis of its different mRNAs. The sequenced gene is the only active gene in human-rodent fibroblastic somatic hybrids, while the other aldolase A-related sequences are inactive. S1 mapping and primer extension analysis enabled us to demonstrate that three promoter regions were implicated in the initiation of different aldolase A mRNAs, differing only in their 5' non-coding extremities. A distal promoter, N (non-specific), governs the synthesis of a 5' non-coding region of 142 bases composed of two exons, N1 and N2, which are found in a variety of tissues. A median promoter, M (muscle), is only active in skeletal muscle, and initiates the transcription by a 5' non-coding exon of 45 bases. Finally, a proximal promoter, H (housekeeping), contained in a "G + C-rich island", permits transcription of three colinear mRNAs containing 172, 126 or 112 bases of 5' non-coding sequence; their expression seems ubiquitous. These three promoters are arranged in 1.5 X 10(3) base-pairs of DNA. Homologies between rat and human genomic sequences and the absence of homology between promoters or 5' non-coding exons of the same species exclude a recent duplication of the promoter regions.