The octamer motif ATGCAAAT is recognized indistinguishably by two mammalian transcription factors: one that is expressed ubiquitously and referred to here as Oct-1, and another, Oct-2, that is expressed in lymphoid cells. We report the cDNA cloning of the human oct-1 gene, which encodes Oct-1, by screening lambda gt11 recombinant phage in situ for octamer motif-specific DNA binding. One lambda gt11 recombinant expressed a beta-galactosidase-octamer-binding fusion protein with a DNA-binding specificity indistinguishable from human HeLa cell Oct-1 protein. As expected for a ubiquitously expressed protein, Oct-1 mRNA is expressed in all five human and two mouse cell lines tested. Polyclonal rabbit antiserum raised against the beta-galactosidase fusion protein shows that the DNA-binding domains of Oct-1 and Oct-2 proteins are related antigenically. Deletion analysis of the 743-amino-acid-long oct-1 open reading frame shows that the DNA-binding activity lies within a central highly charged domain of 160 amino acids. Comparison of the Oct-1 and Oct-2 sequences reveals that this domain is nearly identical between the two proteins. Highly similar domains are also present in the pituitary-specific transcription factor Pit-1 and the Caenorhabditis elegans unc-86 cell lineage gene product (see Herr et al. 1988). Within this shared POU (Pit-1, Oct-1 and Oct-2, unc-86) domain (pronounced 'pow') lie two subdomains: a POU-related homeo box and a POU-specific box. The Oct-1 protein is unique among the POU-related proteins and other homeo box proteins because it is expressed ubiquitously.