Different forms of glucose-6-phosphate dehydrogenase (G-6-PD) have been described in different tissues. Moreover, the directly determined amino acid sequence amino end of the red cell enzyme does not exactly match the sequence deduced from cDNA isolated from HeLa cells or lymphoblasts. We have therefore investigated the sequence of cDNA from sperm, granulocytes, reticulocytes, brain, placenta, liver, lymphoblastoid cells, and cultured fibroblasts. A novel human cDNA, which has extra 138 bases coding 46 amino acids, was isolated from a lymphoblastoid cell library. Sequencing of genomic DNA amplified by the polymerase chain reaction (PCR) revealed that the extra sequence was derived from the 3'-end of intron 7 by alternative splicing. This longer form of mRNA was also detected in sperm and granulocytes. Sequence analysis using PCR-amplified cDNA revealed that the 5'-end of the coding sequence of G6PD mRNA in reticulocytes is identical to those in other tissues.