Genetic studies were performed to examine the role of eukaryotic dnaJ protein, Ydj1p, in the regulated activation of human androgen receptor (hAR) after heterologous expression in Saccharomyces cerevisiae. Hormone-dependent activation of hAR was measured as a function of lacZ reporter gene expression, which was defective in ydj1-151 and ydj1-2(Delta) null mutant strains compared to the wild type. This defect was not due to receptor misfolding, since hAR in both wild type and mutant strains had a similar capacity to bind hormone. The target for Ydj1p action was determined to be the hAR hormone binding domain since an N-terminal fragment lacking this region was constitutively active in both wild type and ydj1-151 mutant strains. These data correlate hormone dependence of hAR activation with a requirement for Ydj1p function and are consistent with a role for dnaJ proteins in signal transduction by steroid hormone receptors.