POU domain proteins interact positively or negatively with steroid hormone receptors, depending on the precise array of these and other factors assembled on target gene promoters. Octamer transcription factor 1 (Oct-1), a ubiquitous POU factor, is implicated in androgen induction of the mouse sex-limited protein (Slp) gene based on protein-DNA interaction studies. However, direct evidence for a role of Oct-1 in the hormone response has been difficult to obtain. Brain 1 (Brn-1), another POU factor, is more tissue-specific, expressing in brain and also in kidney, which is a major site of Slp synthesis. We compared the interaction of the androgen receptor (AR) with Oct-1 and Brn-1 to reveal the more likely candidate for regulation of Slp. In transfection, addition of either Oct-1 or Brn-1 reduced AR activation, regardless of the presence of an octamer-like sequence in the enhancer, suggesting interference was indirect. However, when the octamer-like element was changed to a consensus octamer site, Brn-1, but not Oct-1, strongly enhanced androgen activation. This correlated with Brn-l's preference for the consensus octamer sequence in DNA binding assays. Direct interaction of AR with glutathione-S-transferase-(GST)-fused Oct-1 was DNA-dependent, while Brn-l-AR association was not. Chimeric Brn-1 and Oct-1 POU domains demonstrated that the DNA-dependent AR interaction relied on the origin of the POU homeodomain. However, in the context of full-length Brn-1 and Oct-1 chimeric proteins, the POU homedomain was not sufficient to confer the distinct behaviors of these factors in vivo, but instead revealed the importance of an N-terminal transactivation domain in Brn-1. These results demonstrate that functional interaction of Oct-1 and Brn-1 with AR is determined by the precise sequence of the octamer binding site, and by differential interaction of the POU factors with AR and other components of the transcriptional machinery.