Endothelial nitric oxide synthase (eNOS or NOS3) is the main responsible for nitric oxide (NO) production in vascular system and different polymorphisms have been identified in epidemiological studies. Trying to test the eNOS genetic variation in general populations we studied the 27-bp VNTR in intron 4 and G894T substitution in exon 7 markers in 6 Western Mediterranean populations (3 from Iberian Peninsula, 1 from North Africa, and 2 from Sardinia) and a sample from Ivory Coast. The VNTR frequencies in Western Mediterranean and Ivory Coast fit well into the ranges previously described for Europeans and Sub-Saharans respectively, and a typical African allele has been detected in polymorphic frequencies in the Berber sample. The G894T substitution presents the highest frequencies described for the T allele in the North Mediterranean populations. Linkage disequilibrium is present between both markers in all populations except in the Ivory Coast sample. The variation found for these polymorphisms indicates that they may be a useful tool for population studies even at microgeographical level.