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1. Human high-density lipoproteins HDL2 (d = 1.068-1.125) and HDL3 (d = 1.125-1.210) doubly labelled with [³H]cholesterol/cholesteryl ester and with [acyl-14C]phosphatidylcholine were further incubated with phospholipases. Highly purified phospholipase A2 from Crotalus adamanteus allowed gradual degrees of lipolysis (30-90%) on both HDL2 and HDL3. Moderate phospholipid hydrolyses were achieved using hepatic triacylglycerol lipase, partially purified from post-heparin plasma. Moreover, the latter enzyme seemed to exert a lysophospholipase activity, acting on the 2-acyl-sn-glycero-3-phosphocholine generated. A purified sphingo-myelinase C from Staphylococcus aureus was also used and completely hydrolysed HDL sphingomyelin. 2. After incubation, doubly labelled HDL2/HDL3 were reisolated in their appropriate density interval. In the presence of albumin, which bound most of the lipolysis products, phospholipolysis induced a phospholipid depletion of the particles and a heterogeneous partition of all HDL2 constituents between the HDL2 and HDL3 density intervals. Radioactivity distributions correlated with mass movements. The 'HDL3-like' particles isolated after HDL2 lipolysis were twice as rich in cholesterol as plasma HDL3. No loss of apoprotein A1 was recorded due to phospholipolysis. In the absence of albumin, the density distributions of HDL2 or HDL3 constituents were unaffected by phospholipolysis, the products of lipolysis being reisolated with the stable particles. 3. Control and treated HDL were also reisolated by equilibrium density gradient ultracentrifugation, gel chromatography or by gradient gel electrophoresis. Phospholipase treatment in the presence of albumin induced a shift of the HDL2 or HDL3 whole distribution ... [ABSTRACT FROM AUTHOR]