(1) Various buffer systems for the starch gel electrophoresis of human diaphorase isozymes have been explored. Electrophoresis in a Tris/Borate system at pH 8.6 which includes 70 micron NADH in the gel and cathodal electrode buffers, provides good resolution of the six DIA3 phenotypes previously resolved by isoelectric focusing. (2) The variant genes DIA13, DIA23 and DIA33 occur with frequencies of about 0.76, 0.23 and 0.01 respectively in the English population. (3) The isozymes determined by the least common gene, DIA33, are markedly different from the isozymes determined by DIA13 and DIA23 in their relatively low heat stability, high affinity for Blue Sepharose and slow anodal electrophoretic mobility in buffer systems containing borate. The DIA3 1 and DIA3 2 isozymes are similar to one another in these characteristics.