The regulation of the human corticotropin releasing hormone (CRH) gene is of interest since it mediates the interaction of stress events and inflammatory signals on the hypothalamo-pituitary-adrenal axis. Patients with rheumatoid arthritis (RA) have impaired CRH response to stress so that polymorphisms in the 5' regulatory region of the CRH gene could be responsible for decreased CRH responsiveness. To examine whether patients with RA have polymorphisms of the regulatory region of CRH, we amplified fragments of the 3.7 kb genomic DNA from 5 unrelated patients with RA and 2 controls. DNA sequencing revealed 3 nucleotide variants. These variants have been characterized as a T-->C base substitution located at position 1273 (alleles A1 and A2) and at position 2942 (alleles B1 and B2), and a C-->G base substitution located at position 95 (alleles C1 and C2), respectively, of the Genbank entry x67661. These substitutions lead to the destruction of the recognition site for AflIII (position 1273) and CviJI (position 95), and an introduction of a recognition site for BsmAI (position 2942) restriction enzymes, respectively. Three of 5 patients with RA were heterozygous for all 3 polymorphisms observed, while the 2 polymorphisms at positions 1273 and 2942 were represented in one control only. Post polymerase chain reaction (PCR) restriction fragment length polymorphism was performed to investigate the frequency of the polymorphisms in 99 healthy unrelated Caucasians and 28 members of CEPH (Centre d'Etudes du Polymorphisme Humaine) families. The AflIII, BsmAI, and CviJI polymorphisms cosegregate absolutely in both the controls and the CEPH families. The frequency for allele 1 was 0.869, while that for allele 2 was 0.131, with the following observed genotype frequencies: A1/A1 0.75; A1/A2 0.23; A2/A2 0.02. The T-->C base substitution at position 2942 leads to destruction of a consensus sequence for transcription factor GH-CSE2. These novel polymorphisms should therefore be considered in studies of CRH gene expression and may play a role in the pathogenesis of RA.