A new procedure for the partial purification of Mg2+-dependent, N-ethylmaleimide-sensitive phosphatidate phosphohydrolase (Mg2+-PAP; EC 3.1.3.4) from rat liver cytosol is described, using protein precipitation with MgCl2, gel filtration on Sephacryl S-400, chromatography on DEAE-cellulose and affinity chromatography on calmodulin-agarose. From the parallel change in staining intensity and in the level of the specific activity of enzyme fractions, a relationship between a 90-kDa SDS gel band, identified as the beta-isoform of the 90-kDa heat shock protein, and Mg2+-PAP could be detected.