Hormone‐sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP‐activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca²⁺/calmodulin‐dependent protein kinase II also phosphorylates hormone‐sensitive lipase predominantly at this site, whilst cyclic‐GMP‐dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic‐AMP‐dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone‐sensitive lipase by the cyclic‐AMP‐dependent and cyclic‐GMP‐dependent protein kinases, indicating that site‐2 phosphorylation may have an antilipolytic role in vivo.