Abstract The influence of transcortin-like (TL) binding sites on uptake and distribution of glucocorticoids by dispersed pituitary cells was investigated. TL material, which combines corticosterone (CORT), but not dexamethasone (DEX), was previously found to be present on cell membranes and in cytosol of hypophysis. Exposure of cells at 0° brought about striking differences in steroid binding, as labeled CORT was taken up more rapidly and to a significantly greater extent than DEX. This resulted from a higher concentration of binding sites and not from a difference in binding affinity. At 25 and 37°, while the same relationship was apparent during the early events of steroid interaction with the cell, binding of DEX increased gradually with incubation time and finally exceeded that of the natural steroid. Also, time-course studies on nuclear translocation showed a biphasic pattern, which closely paralleled that of whole-cell binding. Interestingly, treatment of rats with transcortin antiserum caused a decrement of pituitary TL sites, as well as of CORT translocation. We conclude that the TL binder, which is probably of plasma origin, may be actively involved in the process of uptake and cellular distribution of corticosteroids in the pituitary gland.