Rat liver microsomes contain two distinct forms of PtdIns 4-kinase which were resolved by heparin-Sepharose chromatography. One enzyme was identified as the type II PtdIns kinase previously isolated from exocytotic vesicles. The other enzyme, however, was a novel PtdIns 4-kinase isoform with properties differing from any other PtdIns kinase so far characterized. Both kinases were recognized by a monoclonal antibody specific for type II PtdIns 4-kinase, but the novel enzyme was considerably less sensitive to inhibition by adenosine and Ca2+ than type II enzymes, and in addition was specifically inhibited by submillimolar concentrations of dithioerythritol. The presence of a novel PtdIns 4-kinase isoform in rat liver raises the question of whether this enzyme is unique for this organ or whether it has a more widespread distribution but so far has avoided detection.