Glucocorticoid receptors (GCRs) were characterized in sublines of the mouse P1798 lymphosarcoma that are sensitive (S) or resistant (R) to glucocorticoid-mediated apoptosis. Previous work had identified two steroid-binding GCRs in S and R cells: a 97 kDa wild-type GCR in S cells (WT-GCR), and a 45 kDa truncated GCR in R cells (TR-GCR). A third GCR, a 97 kDa nonsteroid-binding GCR (NSB-GCR), was also identified in R cells. Using subcellular fractionation and Western blotting, we now show that in contrast to the WT-GCR which is localized in both the cytoplasm and nucleus of S cells, the NSB-GCR is localized predominantly in R cell nuclei. Moreover, gel filtration chromatography revealed that treatment with 400 mM NaCl and heat did not significantly alter the Stokes radius of the NSB-GCR suggesting that this receptor is not present in a heterooligomeric complex with other proteins. The TR-GCR was localized predominantly in the soluble cytoplasmic fraction but also in the crude membrane fractions of R cell nuclei, suggesting that this receptor is tightly associated with nuclear structures. It was not detected in the soluble nuclear fraction. Unexpectedly, a 45 kDa nonsteroid-binding immunoreactive protein was detected in crude membrane fractions of S cells. These studies describe a complex GCR system in the P1798 lymphosarcoma that necessitates a further consideration of glucocorticoid signaling in S and R cells.