Several viruses have been shown to require calcium for their function, and to bind calcium at specific sites. However, the nature of the calcium binding molecule on viruses has not been established. One possibility is the ubiquitous calcium-binding protein calmodulin. Our studies were designed to determine whether feline leukemia virus contained calmodulin. Accordingly, we tested purified feline leukemia virus for the presence of calmodulin-like activity. The virus, like authentic calmodulin, activated cyclic AMP phosphodiesterase. The ability of the virus to activate the enzyme was blocked in the presence of the known calmodulin inhibitors trifluoperazine and W-7. This indirect evidence for the presence of calmodulin was confirmed by radioimmunoassay. Several other retroviruses were also tested using radioimmunoassay and found to contain calmodulin. Our results indicate that the calcium binding site in retroviruses may be calmodulin.