Different methods of incorporating esterified [3H]cholesterol into human high density lipoproteins (HDL) have been assessed in terms of their influence on the subsequent rate at which esterified [3H]cholesterol was transferred from HDL to other plasma lipoproteins in 37 degrees C incubations containing tracer amounts of the labelled preparations added to aliquots of a common pool of unlabelled human plasma. Two basic methods were used to label the HDL esterified cholesterol: (a) by the action of lecithin: cholesterol acyltransferase in incubations of plasma containing exogenous [3H]cholesterol and (b) by exchange in incubations of plasma containing preparations of prelabelled LDL. The exchange labelling approach was also used to examine the effects of various HDL compositional changes on the subsequent behaviour of its esterified [3H]cholesterol. It was found that the source of the label, per se, whether from the esterification of exogenous [3H]cholesterol or from exchange with labeled LDL, had no influence on the subsequent behaviour of HDL esterified [3H]cholesterol. However, modification of the HDL composition during the labelling process had profound effects. For example, in preparations in which the esterified cholesterol content of the labelled HDL was increased by the action of lecithin: cholesterol acyltransferase, there was a reduced rate of transfer of esterified [3H]cholesterol out of the HDL fraction in subsequent incubations of the labelled HDL with fresh, unlabelled plasma. Conversely, when the esterified cholesterol content of the labelled HDL had been decreased by pre-incubation with Intralipid or very low density lipoproteins (VLDL), the subsequent rate of transfer of esterified [3H]cholesterol out of the HDL fraction was increased markedly. It has been concluded that to obtain a biologically valid, labelled tracer of human HDL esterified cholesterol, the labelling should be achieved with minimal modification to the HDL composition. This means labelling by exchange in incubations in which lecithin: cholesterol acyltransferase is inhibited in plasma from which VLDL has been removed.