Gram-negative, ionophore-resistant ruminal bacteria and Gram-positive, ionophore-sensitive species bound similar amounts of [14C]lasalocid, but neither group bound large amounts of [14C]monensin. Membrane vesicles also bound more lasalocid than monensin (P < .05). The binding was first-order at low cell or vesicle concentrations and saturable at high cell or vesicle densities. Streptococcus bovis was inhibited by both monensin and lasalocid (5 microM), but cells that were re-incubated in medium lacking ionophore grew rapidly. Lasalocid-treated cells grew very slowly when they were resuspended in fresh medium. Based on these results, it seemed that lasalocid had a higher affinity for bacterial membranes than monensin. Mixed bacteria, however, bound nearly equal amounts of [14C]monensin and [14C]lasalocid (P > .05). Monensin binding was greatly reduced when the mixed ruminal bacteria were pretreated with Tris+EDTA (P < .05), but Tris+EDTA did not affect the binding of lasalocid. Mixed ruminal protozoa always took up more lasalocid than monensin (P < .05), but feed particles bound equal amounts of [14C]lasalocid and [14C]monensin (P > .05). Based on the binding capacity of mixed ruminal bacteria, ruminal protozoa, and feed particles, there would be little free ionophore in ruminal fluid.