The sensitivity of lymphosarcoma P1798 to glucocorticoids varied as a function of growth conditions. Cells grown in the ascitic fluid were very sensitive to cortisol inhibition of tritiated thymidine ([3H]dThd) incorporation. When ascites cells were inoculated sc into BALB/c mice receiving daily injections of 2 mg cortisol, tumors did not form. However, as tumors grew subcutaneously in control mice, glucocorticoid sensitivity decreased to the arrested by cortisol injection. Cortisol also did not inhibit incorporation of [3H]dThd in cells prepared from large subcutaneous tumors. Measurement of cytoplasmic receptors in cell-free extracts revealed that both ascites and subcutaneous tumor cells contained about 11-12 x 10(4) glucocorticoid binding sites per cell. Receptors in ascites cells had a higher affinity for glucocorticoids than did receptors in subcutaneous cells, which indicated that sensitive and resistant cells contain chemically different classes of receptors. Loss of sensitivity occurred as tumors attained a diameter greater than approximately 1.5 cm. The reproducibility of this transition did not appear to be consistent with a random mechanism for loss of glucocorticoid sensitivity in vivo. Thus the acquisition of resistance by lymphosarcoma P1798 in vivo was concluded to be nonmutational and probably resulted from differentiational alteration in gene expression.