The effects of the antibiotic acetylspiramycin (ASPM) on lymphocyte function were studied in vitro and in vivo. When added to lymphocyte cultures in vitro, ASPM inhibited splenic lymphocyte transformation induced by phytohemagglutinin (PHA), lipopolysaccharide (LPS) and antigen. It also depressed production by spleen cells of the lymphokine inducing procoagulant activity in mouse macrophages. Spleen cells from mice given ASPM orally showed enhanced responses to PHA, but normal responses to LPS. The capacity to produce lymphokine was increased early after oral ASPM and slightly decreased after prolonged administration. Oral ASPM had no effect on the production of antibodies and a very slight enhancing effect on the development of delayed-type hypersensitivity to sheep red blood cells.