Heritability analyses were performed with clinical chemistry data collected on 360 twin pairs of white, middle-aged male veterans during the second examination of the NHLBI Twin Study, a multicenter study of cardiovascular disease risk factors. Significant genetic variability was present for albumin, alkaline phosphatase, blood urea nitrogen, 1-hr postload glucose, phosphorus, total protein, and uric acid. Calcium and aspartate aminotransferase had significantly different means by zygosity, which precluded further analysis. Total bilirubin and lactate dehydrogenase did not show evidence for genetic variation at this examination. Comparisons are made to results from similar twin studies and the first examination of the NHLBI Twin Study. Heritability estimates for phosphorus and blood urea nitrogen exhibited marked stability across studies, while heritability estimates for total bilirubin, total protein, and uric acid decreased in older study populations. The heritability of 1-hr postload blood glucose decreased from 0.88 at the first NHLBI examination to 0.52 at the second one. Interpretation of these results requires consideration of possible selection biases, methodologic and demographic issues, and the view that for some clinical chemistries, biological aging along with prolonged environmental exposures may alter the amount of phenotypic variation explained by the additive effect of genes alone.