Multiple protein 4.1 isoforms are originated by alternative pre-mRNA splicing, differential use of two translation initiation sites, and posttranslational modifications. The complexity of alternative splicing events suffered by the 4.1 pre-mRNA makes necessary the direct cloning of 4.1 full-coding cDNA sequences to ensure that the encoded 4.1 proteins are naturally occurring isoforms. We have approached this point by reverse transcription-polymerase chain reaction techniques using RNA from the nucleated human Molt-4 T-cell line as a starting template. Molecular cloning of 4.1 cDNAs using the second translation initiation codon has allowed us to identify two 4.1 isoforms, designated 4.1H and 4.1I, which are differentially targeted to the nucleus (4.1H) and the cytoplasm (4.1I). These two isoforms differ only in the inclusion (4.1H) or exclusion (4.1I) of 21 amino acids encoded by exon 16. A cluster of basic amino acids, KKKR, generated by joining of the sequences encoded by the constitutive exon 13 and the alternative exon 16, is necessary for the nuclear targeting of 4.1H, as demonstrated by site-directed mutagenesis analysis. Immunofluorescence microscopy and biochemical studies indicate that 4.1H belongs to the group of nuclear 4.1 proteins that are distributed diffusely throughout the nucleoplasm and that are extractable in 0.5% Triton X-100. This is the first demonstration of differential nuclear targeting by the presence of an alternative domain, among naturally occurring protein 4.1 isoforms.