Molecular forms of sphingomyelinase and phosphodiesterases from lymphocytes- and Epstein-Barr virus-transformed lymphoid cell lines were separated by preparative electrofocusing in granulated gels. In either type of cell derived from normal individuals, sphingomyelinase focused as a single peak (pI = 5.60 +/- 0.1) while phosphodiesterases hydrolyzing bis(4-methylumbelliferyl)phosphate and bis(4-methylumbelliferyl)diphosphate separated into seven and three molecular forms respectively; one of the latter showed sphingomyelinase as well as phosphodiesterase activities. Lymphoid cell lines derived from patients with Niemann-Pick disease, types A or B, were practically devoid of sphingomyelinase activity; this was not so for the phosphodiesterases which focussed essentially as normal. The protein peak, which in normal cells contained the three activities, had phosphodiesterase but no sphingomyelinase activity in the Niemann-Pick cells. In normal cells, sphingomyelinase and phosphodiesterase activities of this peak showed different responses to heating and several effectors. These data suggest that in lymphoid cell lines, which are a useful model for studies of Niemann-Pick disease, sphingomyelinase and phosphodiesterases are subject to separate genetic coding and that the latter activities are not a reliable measure for diagnosing Niemann-Pick disease.