The outside-inside translocation rate and transmembrane equilibrium distribution, at 37 degrees C, of 16 different amphiphilic spin-labeled phospholipids have been determined in human erythrocytes. The transmembrane distribution was assessed by bovine serum albumin extraction of the spin-labels present in the outer monolayer. Within 15 min, more than 90% of the phosphatidylserine analogue was found in the inner monolayer; the equilibrium distribution of phosphatidylethanolamine spin-label was approximately 85-90% inside, with a half-time for translocation of approximately 50 min. In contrast, phosphatidylcholine reached a distribution corresponding to approximately 30% of the labels inside with a half-time of approximately 8 h, and only traces of sphingomyelin were found in the inner monolayer after 16 h. Thus, the spin-label analogues distributed themselves like endogenous phospholipids in red cells with a spontaneous segregation between the amino lipids and the choline-containing phospholipids. Progressive methylation of the amine group of phosphatidylethanolamine resulted in a stepwise decrease of the specific transport; modification of the beta-carbon of the serine also decreased the efficiency of the rapid translocation without abolishing it. Phosphatidyl-propanolamine was not transported. Substitution of the glyceride group by a ceramide abolished the rapid outside-inside translocation even with a molecule bearing a serine head group. Also it was found that esterification of the sn-2 position of the glycerol component was necessary for a rapid translocation since lysophosphatidylserine was only slowly transported from outside to inside.(ABSTRACT TRUNCATED AT 250 WORDS)