Mycobacterium leprae synthesizes large quantities of a specific phthiocerol-containing phenolic glycolipid in vivo. We have shown earlier that viable M. leprae readily incorporates radiolabeled palmitic acid into phenolic glycolipid I when residing in cultured macrophages in vitro and that this process is inhibited by the antileprosy drug rifampin. In the present paper we report that application of this observation to the rapid evaluation of over 25 antimicrobial agents for potential antileprosy activity in vitro. All the known antileprosy drugs rifampin, dapsone, clofazimine, and ethionamide inhibited phenolic glycolipid I synthesis. Rifabutin, a spiropiperidyl derivative of rifamycin, also reported to be active in the mouse model, was very effective. Interestingly, the macrolides erythromycin, clarithromycin, and roxithromycin were also found to be active in this system, while D-cycloserine and other cell wall synthesis inhibitors showed no effect. Many of the compounds found to be active in this system have been reported to be effective in vivo in mice. This correlation lends support to the feasibility of using phenolic glycolipid I synthesis for the rapid evaluation of new drugs against leprosy.