Numerous eukaryotic upstream promoter and enhancer regions contain a functional octamer sequence ATGCAAAT. We have examined the interactions between an octamer binding protein isolated from HeLa cells and the SV40 and immunoglobulin heavy-chain (IgH) gene enhancers. A partially purified octamer binding activity forms a single complex with the IgH enhancer octamer in a gel retardation assay, but two complexes with a SV40 enhancer fragment containing a single 72-bp element. By using point mutants and both dimethyl sulfate and diethyl pyrocarbonate modification interference assays, we show that the SV40 complexes result from binding of a factor to the octamer-related sequence ATGCAAAG (Octa1) and to an adjacent previously unidentified octamer-related sequence ATGCATCT (Octa2). The base-specific interactions with Octa1 and Octa2 differ; chemical modifications over a 10-bp sequence TATGCAAAGC affect Octa1 binding whereas Octa2 binding is affected by modifications spanning a 13-bp sequence ATGCATCTCAATT in which the octamer-like sequence is not centered. The octamer binding activity has been purified extensively by a DNA affinity precipitation procedure and SDS-polyacrylamide gel electrophoresis. The purified protein, OBP100, has an apparent molecular weight of 100 kD and binds both SV40 Octa1 and Octa2, as well as the IgH enhancer. The distinct interactions of OBP100 with the differently sized Octa1 and Octa2 binding sites suggest remarkably flexible sequence recognition between OBP100 and its binding sites.