Rat adipose hormone-sensitive lipase-mediated release of fatty acids from triglycerides was studied in three model systems: i) cultured preadipocytes containing polyunsaturated fatty acid-enriched triglyceride; ii) perfused epididymal fat pads; and iii) in vitro incubations of crude preparations of hormone-sensitive lipase with synthetic triglyceride-analogues as substrates. We found that cultured preadipocytes challenged with 10 microM norepinephrine tended to release more omega 6 and omega 3 polyunsaturated fatty acids than saturated fatty acids. Fat pads perfused with 10 microM norepinephrine preferentially released arachidonate and alpha-linolenate but tended to retain oleate and linoleate. Finally, crude preparations of hormone-sensitive lipase released from the triglyceride-analogue substrates alpha-linolenate twice as fast as oleate. We conclude that rat adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides. We suggest that this may be a mechanism by which these fatty acids are kept from being trapped in fat depots and maintained in the circulation.