Human small cell lung carcinomas (SCLCs) frequently express the adrenocorticotrophin precursor gene proopiomelanocortin. Glucocorticoids usually fail to inhibit this ectopic adrenocorticotrophin production, in contrast to their effects in the pituitary. We have shown three human SCLC cell lines to be globally resistant to glucocorticoid action; in two of these lines this occurs despite the presence of glucocorticoid receptors (GR+). Accordingly, we have cloned and sequenced the GR coding region from one of these two GR+, SCLC cell lines, COR L24, and identified compound heterozygous mutations. One allele had a single nucleotide substitution of A to G in the NH2-terminal domain, which altered Asp to Ser at amino acid 363. The other allele contained a trinucleotide insertion at the 5' boundary of exon 4, which introduced an additional amino acid, Arg453, between the two zinc fingers of the DNA binding domain. In cotransfection studies using the glucocorticoid responsive mouse mammary tumor virus-luciferase Ser363 did not alter receptor function. In contrast, Arg453 encoded a GR with 48% Vmax activity compared to wild-type receptor (P < 0.001), with an unchanged EC50. Thus, GR mutations may contribute to the glucocorticoid-resistant phenotype of GR+ COR L24 cells, which could confer survival advantage to this highly malignant neuroendocrine tumor.