The prototypical erythrocyte membrane skeletal protein 4.1 (HGMW-approved symbol EPB41), here designated 4.1R, is encoded by a large, complexly spliced gene located on human chromosome 1p32-p33. In this paper we report evidence for a second 4.1 gene, 4.1G (HGMW-approved symbol EPB41L2), which maps to human chromosome 6q23 and is widely expressed among human tissues. The complete nucleotide sequence of 4.1G cDNA predicts a 113-kDa protein that exhibits three regions of high homology to 4.1R, including the membrane binding domain, the spectrinactin binding domain, and the C-terminal domain. Interspersed among the shared domains are unique sequences that may define functional differences between 4.1R and 4.1G. Specific isoforms of 4.1R and 4.1G exhibit differential subcellular localizations. These results expand the 4.1 gene superfamily and demonstrate that the diverse cellular complement of 4.1 isoforms results from both alternative splicing and expression of distinct genes.