A large family of tissue-specific nuclear proteins interact with the octamer motif ATTTGCAT, a transcriptional regulatory element found in the promoter and enhancer sequences of many genes. As a step towards elucidating the mechanism of this regulation, cDNA clones of the mouse Oct2 protein were isolated. One, called here Oct2b, encodes a larger variant of the previously described Oct2a proteins. The Oct2b cDNA has an insertion of 74 bp close to the 3' end which creates an open reading frame distinct from Oct2a. As a result, the Oct2b protein has a carboxy end which is similar to that of the ubiquitous octamer-binding protein Oct1. Analysis of the Oct2 gene shows that Oct2a and Oct2b are differentially spliced products of the same gene. The insertion in the Oct2b cDNA results from the inclusion of an additional exon in the mRNA which would otherwise reside in an intron sequence of the Oct2a transcript. RNA analysis demonstrates that both Oct2a and 2b mRNAs are most abundant in B-cells but they are also expressed in a variety of tissues including brain, intestine, testis, kidney, as well as in embryos. Interestingly, the ratio of Oct2a and 2b varies among tissues. In situ hybridization studies during mouse embryogenesis show that the Oct2 gene is widely expressed in the developing nervous system. In contrast, expression in the adult brain is confined to very specific areas which include the suprachiasmatic and medial mammillary nuclei, hippocampus, olfactory tract and the olfactory bulb. Oct2 proteins are present in both neuronal and oligodendroglial cells, although they are more abundant in glial cells.