S49, an established line of mouse lymphoma cells, has several characteristics useful for the genetic analysis of glucocorticoid action: (1) a stable pseudodiploid karyotype; (2) an efficient cytolytic effect of glucocorticoids, which appears to follow the same biochemical pathway as steroid hormone action in other systems; (3) appearance of rare steroid-insensitive clones that exhibit stable, heritable resistance to further glucocorticoid treatment; (4) rapid growth in suspension culture and high cloning efficiency in soft agar, allowing facile isolation of variant clones. Two hundred individual steroid-resistant clones of S49 cells have been isolated and analyzed to determine the origin of their resistance. Most of these variant clones (55 %) fail to bind [3H]dexamethasone at levels above background; 70--75 percent bind less than 30 % of the wild-type level. The remaining clones fall into three general groups with respect to the efficiency with which receptors are translocated to the nucleus following dexamethasone treatment: one class transfers less than 10 percent of labeled receptors to the nucleus, another transfers normal amounts, and a third localizes virtually all of the receptors in the nucleus. The four variant phenotypes have been respectively designated r-, receptor activity deficient; nt-, nuclear transfer deficient; d-, deathless (appears normal in binding and nuclear transfer); and nti, increased nuclear transfer. Physical characterization by sucrose gradient sedimentation and gel permeation chromatography reveals that wild-type receptors are approximately 90,000 daltons and nti receptors about 50,000 daltons. The affinities of variant and wild-type receptors for purified DNA in vitro are consistent with their respective nuclear binding characteristics in vivo. Genetic studies with these and other cell lines, combined with recently developed methods for purification and structural analysis of minute quantities of proteins, can provide the level of biochemical resolution required for a fundamental understanding of the molecular mechanism of steroid hormone action.