Heat shock protein 90 (Hsp90) is an abundant molecular chaperone required for the proper function of a selected group of client proteins with key regulatory roles in cellular signalling. Among the best studied client proteins are steroid receptors which undergo step-wise assembly with Hsp90 complexes containing one of four Hsp90-bound tetratricopeptide repeat (TPR) immunophilin cochaperones – FKBP51, FKBP52, cyclophilin 40 (CyP40) and protein phosphatase 5 (PP5). A dynamic assembly cycle promotes the exchange of these cochaperones allowing the receptor ligand-binding domain (LBD) to effectively sample the hormonal environment, with complexes involving FKBP52 helping androgen (AR), glucocorticoid (GR) and progesterone (PR) receptors achieve a high affinity hormone-binding state with increased transcriptional activity. Progress in crystallographic analysis, making available structures for ligand-bound receptor LBDs, the TPR cochaperones and Hsp90, has provided important insights into the mechanisms associated with the dependence of the steroid receptor LBD on Hsp90 molecular chaperone machinery. At the same time, application of yeast two-hybrid analysis using the receptor LBD as a target has revealed additional Hsp90 TPR cochaperones such as GCUNC-45 and aSGT, that function at intermediate stages of the steroid receptor/Hsp90 pathway to regulate the activity of specific receptors.