POU homeodomain proteins are important regulators of ubiquitous as well as tissue-specific transcription. These factors include the broadly expressed octamer motif-binding protein Oct-1 and the related cell-specifically expressed protein Oct-2. These two proteins differ in the types of octamer motif-containing promoters they preferentially activate; Oct-1 can activate RNA polymerase II transcription from a small nuclear RNA promoter better than Oct-2, which can better activate an mRNA-type promoter. We describe a variant Oct-1-encoding cDNA resulting from two separate alternate splices of the human oct-1 primary transcript; these alternate splices were present in all cell lines tested. This cDNA encodes an amino-terminally and carboxyl-terminally truncated form of Oct-1, called Oct-1B, which retains the DNA-binding POU domain and acquires a unique 12-amino acid carboxyl-terminal extension. In a transient expression assay, Oct-1B displayed an enhanced ability compared to the larger form of Oct-1 (called Oct-1A in this report) to activate the human histone H2B promoter, an mRNA-type promoter where a natural octamer motif is involved in cell cycle dependent transcription. Thus, the ability of Oct-1 related proteins to activate a natural regulatory target can be influenced by alternative splicing.