The increase of fructose-1,6-diphosphatase activity during culture with calcitriol, which was reported in monocytes, was found not to occur in lymphocytes. Monocytes cultured with calcitriol were accordingly used as more reliable diagnosis of heterozygotes for fructose-1,6-diphosphatase deficiency, instead of mononuclear cells (lymphocyte-fraction-containing monocytes) cultured without calcitriol by a conventional method. Variation of fructose-1,6-diphosphatase values in leukocytes from nine healthy adults was smallest in monocytes cultured with calcitriol, among four different experimental conditions: monocytes cultured with or without calcitriol and mononuclear cells cultured with or without calcitriol. Both parents of two sisters with fructose-1,6-diphosphatase deficiency were successfully confirmed as carriers of fructose-1,6-diphosphatase deficiency by this method. However, confirmation by the conventional method using mononuclear cells cultured without calcitriol was possible only in the father, not in the mother. Thus, the new method using monocytes cultured with calcitriol seems more reliable for detecting heterozygotes for fructose-1,6-diphosphatase deficiency.