To explore the hypothesis that susceptibility to cerebral malaria is influenced by genetic variation in endothelial nitric oxide synthase (eNOS), we genotyped three commonly defined polymorphic loci of eNOS, Glu(298)-->Asp, intron 4 variable number of tandem repeat region, and T-786-->C, in 244 patients (mean age, 36.2 years) with mild malaria and 194 patients (mean age, 35.6 years) with severe malaria belonging to same ethnic group in Orissa, an eastern Indian state. We found that there was an association of the Glu(298)-->Asp substitution (P = 0.0037; odds ratio, 1.95; 95% confidence interval, 1.2 to 3.0) and a single unique haplotype defined by "C-b-Asp" (P(corrected) = 0.0024) for protection against cerebral malaria. Further, the median plasma level of nitrite-nitrate was found to be increased in individuals with the Glu(298)-->Asp substitution and was significantly higher in the mild malaria group (P <or= 0.0001), but the increase was not significant in the severe malaria group (P = 0.0528). These findings suggest that the Glu(298)-->Asp substitution and the "C-b-Asp" haplotype may enhance eNOS expression and NO production, which leads to protection against cerebral malaria. These findings may increase our understanding of the pathogenesis of malaria.