Since measurement of lysophosphatidate phosphatase activity is important in studies of tumorigenesis, we attempted to develop a simpler alternative to the more complex methods currently available. Measuring the phosphate released would permit use of the same method for a variety of phosphatases with physiological substrates, many of which are nonchromogenic. The Malachite green method of K. Itaya and M. Ui (1966, Clin. Chim. Acta 14, 361) has adequate sensitivity for quantitating phosphatase activity in biological samples. In samples with high endogenous phosphate concentrations pretreatment with 50 mg Dowex 1 × 10 (100-200 mesh, OH- form) usually permitted reliable determination of phosphatase activity. For 34 consecutive runs the mean relative difference [(phosphorus activity - vitamer activity)/phosphorus activity] obtained from the simultaneous measurement of both the phosphate released and the corresponding organic product (pyridoxal and pyridoxine) was -0.03 ± 0.09. The within run and between run coefficients of variation (three runs of four to five replicates) were 0.05 and 0.04, respectively. Pyridoxine 5′-phosphate hydrolase activity (pH 10) in cultured skin cells (normal and cancerous) ranged from 2 to 12 nmol phosphorus/min·mg protein. Lysophosphatidate phosphatase activity (pH 7.4) ranged from 3 to 14 nmol phosphorus/min·mg protein. The current approach permits the measurement of phosphatase activity with a single method using a variety of substrates and incubation conditions. © 2001 Academic Press.