Transfer of cholesteryl ester (CE) between lipoproteins is mediated by a plasma glycoprotein called cholesteryl ester transfer protein (CETP) which plays an important role in reverse cholesterol transport between peripheral tissues and the liver. Adipose tissue is a major site of cholesterol storage and lipoprotein interactions. CE transfer activity in cultured adipose tissue from several species was analyzed by using a CE transfer assay in vitro. Significant amounts of CE transfer activity were detected in conditioned media of cultured adipose tissues from human, hamster and transgenic mice expressing CETP. The amount of CE transfer activity released from cultured adipose tissue varied among species and adipose regions. Monoclonal antibodies against human CETP (subclone TP2) inhibited CE transfer activity released from cultured adipose tissue of hamsters and CETP transgenic mice. CE transfer activity was also assayed in the aqueous phase of adipose tissue homogenates. Accumulation of CE transfer activity in the medium was associated with a reciprocal decrease in adipose tissue activity positively correlated to the amount of CETP mass in the conditioned medium and the aqueous phase of tissue homogenates from cultured hamster adipose tissue. The results of the present study indicate that the measurement of CE transfer activity in adipose tissue reliably characterizes the secretion and storage of CETP in adipose tissue.