We studied the mechanism of POMC gene expression in human nonpituitary tumors that is responsible for the ectopic ACTH syndrome. All tumors contained a 1200-nucleotide (nt) POMC mRNA species identical to that in normal and tumoral pituitaries. In two of six nonpituitary tumors, equivalent amounts of a larger, ca. 1450-nt POMC mRNA species were also present. S1 mapping studies with probes encompassing the three exons of the gene revealed that this larger POMC mRNA species was 5' extended; the other regions were identical to that in the 1200-nt POMC mRNA. In order to analyze the 5'-end of the larger POMC mRNA species, a genomic clone starting at 3.0 kilobases upstream from the usual (pituitary) cap site was obtained, and single-stranded DNA probes were used for S1 mapping studies. They showed several upstream start sites of transcription located at -369, -217, and -108. Analysis of the human genomic sequence showed TATA and GC box-like motifs preceding the -369 and -217 sites and a GC-rich region preceding the -108 site. S1 mapping with a DNA probe, encompassing exon 1 and 93 nt of its 5'-flanking region, allowed quantitative determinations, which showed that the 5'-extended POMC mRNA species accounted for variable proportions of the overall POMC transcripts in different tissues: 0.3% or less in two normal pituitaries, 0.5-3% in five tumoral pituitaries, and up to 35% and 40% in two of six nonpituitary tumors. These results show that variable modes of human POMC gene expression are induced by upstream promotors whose relative activities appear increased in some nonpituitary tumors.(ABSTRACT TRUNCATED AT 250 WORDS)