We have identified an alternatively spliced form of the human beta 1 integrin subunit, beta 1S. The beta 1S mRNA is expressed in human platelets, HEL and K562 erythroleukemia cell lines, and THP1 monocytic and HL60 promyelocytic cell lines. It is undetectable in peripheral blood lymphocytes and cultured umbilical vein endothelial cells at early passages. The beta 1S cDNA encodes a new cytoplasmic domain distinct from the previously reported alternative cytoplasmic domain of the beta 1 subunit. The sequence reveals the presence of an insert of 116 nucleotides which produces a frame shift in the previously reported 3' end of the beta 1 integrin subunit and codes for a unique 48-amino acid COOH-terminal sequence. An antiserum prepared against a synthetic peptide generated from the deduced sequence of the beta 1S cytoplasmic domain immunoprecipitated an HEL cell surface molecule that comigrated with the usual beta 1 subunit in sodium dodecyl sulfate electrophoresis. The immunoprecipitation indicated that beta 1S constitutes a minor portion of total beta 1 subunit in these cells. This variant beta 1 cytoplasmic tail may modulate integrin affinity and may provide additional modes for the transduction of extracellular signals and modulation of cytoskeletal organization by beta 1 integrins.