The human T-cell- or lymphocyte-specific gene, lck, encodes a tyrosine kinase and is a member of the src family. In this report we demonstrate that there are two classes of human lck transcripts (types I and II), containing different 5'-untranslated regions, which are expressed from two distinct promoters. No apparent sequence similarity was observed between the 5'-flanking regions of the two promoters. The expression of lck in human T-cell leukemia and carcinoma cell lines and in human peripheral blood T lymphocytes was examined by S1 nuclease and primer extension mapping and by Northern (RNA) blot analysis of total cellular RNA. The following results were obtained. (i) Two RNA start sites in the downstream promoter were used to generate type I transcripts. (ii) The major human type I start site has not been described for the mouse. (iii) At least five RNA start sites in the upstream promoter were used to generate type II transcripts. (iv) In T cells and in two colon carcinoma cell lines, type II transcripts were present in higher amounts than type I transcripts. (v) In T cells treated with phytohemagglutinin, tetradecanoylphorbol acetate, and cyclosporin A, the modulation of lck expression was associated primarily with changes in levels of type II transcripts. The above results suggest that the two human lck promoters are utilized differentially and may be regulated independently during certain physiological states.