The atrial natriuretic peptide (ANP) gene was, though inconclusive, implied to be etiologically related to stroke in rats and recently in humans. The present study tested the candidacy of ANP for stroke susceptibility by a combination of molecular genetic approaches. First, we undertook an association study using a reported ANP variant, G664A, in two case-control panels independently collected, which involved 970 Japanese subjects. Second, we compared the rat ANP gene sequences and neighboring marker alleles among stroke-prone SHR (SHRSP), normal SHR and WKY of an original inbred colony and we also compared brain ANP expression between SHRSP and normal SHR. In humans, we found no significant association between the 664A variant and stroke in the studied population. In rats, 21 polymorphic sites were identified by direct sequencing of 2170-bp ANP fragments, from which two distinct alleles, SHRSP- and WKY-types, were inferred. From a genealogical point of view, our data indicated that an SHRSP-type allele could not play a determinant role in stroke-proneness. Overall results did not support the disease relevance of ANP, disagreeing with previous reports. Thus, considerable caution should be taken when one attempts to transfer findings in the animal model to humans.