The lck gene, which encodes the lymphoid cell-specific tyrosine protein kinase p56lck, is expressed from two widely separated promoters. The proximal promoter gives rise to a type I lck transcript, and the distal promoter gives rise to a type II transcript. We found that the ratio of the two transcripts changed during T-cell maturation. Type I lck mRNA was twofold more abundant than the type II transcript in early fetal thymocytes. In the adult, the type I and type II lck mRNAs were present in approximately equal amounts in immature thymocytes expressing the heat-stable antigen. In contrast, there was five- to ninefold more type II lck than type I lck mRNA in more mature thymocytes that did not express the heat-stable antigen and in splenic T cells. This change in relative transcript abundance probably reflects activation of the distal promoter and inactivation of the proximal promoter during T-cell maturation in the thymus. It is possible that the two promoters are regulated by different trans-acting factors whose expression is regulated during T-cell maturation.