The subcellular forms of cytidylyltransferase (EC 22.214.171.124) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97,690 +/- 10,175. Thus, the L-Form appears to be a dimer of the Mr 45,000 catalytic subunit. The f/f degrees value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284,000 +/- 25,000. The H-Form was dissociated into L-Form by incubation of cytosol at 37 degrees C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45,000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45,000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.