Corticotropin-releasing factor (CRF) appears to regulate several physiological systems that display prominent abnormalities in Zucker fatty (fa/fa) rats, including the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and feeding behavior. Moreover, central administration of CRF ameliorates the obese phenotype. In light of these observations, the gene for CRF is a plausible candidate for the defective gene in the Zucker fatty rat. We report here the use of molecular genetic linkage analysis to test the hypothesis that fa is a mutant allele of the CRF gene. A restriction fragment length polymorphism for CRF between Zucker (13M) and Brown Norway (BN) DNA allowed us to examine segregation of 13M and BN CRF alleles relative to fa in 58 obese (fa/fa) F2 progeny of a 13MBN fa/+F1 intercross. If fa = CRF, all animals homozygous for the fatty mutation should be homozygous for the 13M CRF allele. However, only 10/58 fa/fa animals were homozygous for the 13M CRF allele, indicating that fa and CRF are not allelic. Thus, although CRF may be important in the physiology of Zucker rat obesity, fa is not a CRF mutation. Using a mouse C57BL/6J Spretus F1 x C57BL DBA/2J F1 intercross, we were able to demonstrate that the mouse CRF gene is linked to the carbonic anhydrase II (Car-2) gene on mouse chromosome 3, in a region of synteny-homology with rat chromosome 2. Thus the rat CRF gene is probably located on chromosome 2.