The Hsp90 and Hsp70 molecular chaperones play important roles in the folding and proper functioning of diverse cellular proteins, including transcriptional regulators and protein kinases. In yeast, several transcriptional regulators and protein kinases are known to be substrates for Hsp90 and Hsp70 molecular chaperones. The yeast heme activator protein Hap1 promotes transcription of many genes in response to heme. It requires Hsp90 and Hsp70 molecular chaperones for its activity to be precisely regulated by heme concentration. The mechanism by which molecular chaperones promote heme regulation of Hap1 activity is distinct from the mechanism by which molecular chaperones promote steroid signaling. Hsp70 and Hsp90 molecular chaperones act separately to promote Hap1 repression in heme-deficient cells and heme activation of Hap1 in heme-sufficient cells. Likewise, distinct Hap1 elements or domains act to mediate Hap1 repression and heme activation separately. In this review, we summarize the current knowledge about the molecular mechanism governing heme regulation of Hap1 activity, and we compare this mechanism to the molecular mechanism by which Hsp90 and Hsp70 molecular chaperones promote the regulation of glucocorticoid receptor, the most extensively studied substrate of Hsp90 and Hsp70 molecular chaperones.