Pyridoxal phosphate influences several properties of steroid hormone receptors in vitro, but its role in vivo has not been clearly established. In an effort to address this issue, we have investigated the in vivo effects of vitamin B6 on the physical properties and biological function of the human glucocorticoid receptor. We demonstrate that vitamin B6 treatment of whole cells in culture produces an alteration in the isoelectric point of the receptor, as well as changes in the steroid and DNA binding capacities. Furthermore, glucocorticoid dependent transcriptional activation properties of the receptor are also altered by modulation of the vitamin B6 status. High concentrations of vitamin B6 suppress activation of transcription, while vitamin deficiency enhances responsiveness to steroid hormone. Together, these studies imply a physiological role for vitamin B6 in glucocorticoid hormone action.