When a membrane suspension prepared from isolated rat brown fat mitochondria was incubated at 37 degrees C for 4 h, a loss of uncoupling protein (UCP) immunoreactivity was observed on Western blots. Analysis of [3H]GDP-binding characteristics to UCP in isolated membranes also showed a significant reduction in Bmax without significant effect on Kd. The loss of UCP was not due to protease contamination from lysosomes or mast cell granules, since loss of UCP was still observed when mitochondria were treated with digitonin to lyse lysosomes prior to membrane preparation and when mitochondria were isolated from rats injected with compound 48/80 to degranulate mast cells. Furthermore, loss of UCP was observed at alkaline pH and was not affected by inhibitors of lysosomal enzymes. Loss of UCP immunoreactivity was markedly reduced when membranes were incubated at 4 degrees C or in the presence of phenylmethylsulfonyl fluoride, but was not influenced by the addition of GDP. Overall, these results indicate the presence of a serine protease within brown fat mitochondrial membranes that may be involved in the breakdown of UCP.