Squirrel monkeys have high cortisol compared to Old World primates to compensate for glucocorticoid resistance. Glucocorticoid resistance in squirrel monkeys may result from mutations in the glucocorticoid receptor (GR) that render it less transcriptionally competent, or expression of the co-chaperone FKBP51 that reduces ligand binding. The goal of this study was to reconcile the contribution of each mechanism. Responsiveness of squirrel monkey GR in COS-7 cells was reduced compared to human GR, but induction of GR activity by maximum dexamethasone concentrations was similar. Also, expression of squirrel monkey FKBP51 reduced responsiveness of both squirrel monkey and human GR in T-REx-293 cells. The EC(50) for dexamethasone was 100-fold higher in cells expressing squirrel monkey GR and excess FKBP51 compared to cells expressing only human GR. Effects of FKBP51 expression and treatment with FK506 were also determined in squirrel monkey SQMK-FP cells that naturally express high levels of FKBP51. Overexpression of FKBP51 in SQMK-FP cells had little effect on GR responsiveness, but treatment with FK506 that blocks the effect of FKBP51 increased GR responsiveness. Thus, glucocorticoid resistance in squirrel monkey cells results from both expression of GRs that are less responsive and overexpression of FKBP51 that further reduces GR responsiveness.