The kinetics and specificity of l-lactate transport into cardiac muscle were studied during a single transit through the isolated perfused rabbit heart using a rapid (15 s) paired-tracer dilution technique. Kinetic experiments revealed that lactate influx was highly stereospecific and saturable with an apparent K_t = 19 ± 6 mM and a V_max = 8.4 ± 1.5 μmol/min per g mean ± S.E., n = 14 hearts). At high perfusate concentrations (10 mM), the inhibitors α-cyano-4-hydroxycinnamate (K_i = 7.3 mM), pyruvate (K_i = 6.5 mM), acetate (K_i = 19.4 mM) and chloroacetate (K_i = 28 mM reduced l-lactate influx, and K_i values were estimated assuming a purely competitive interaction of the inhibitors with the monocarboxylate carrier. The monocarboxylic acids [¹⁴C]pyruvate and [³H]acetate were themselves transported, and sarcolemmal uptakes of respectively 38 ± 1% and 70 ± 8% were measured relative to d-mannitol. Perfusion of hearts for 10–30 min with 0.15 or 1.5 μM glucagon increased myocardial lactate production and simultaneously inhibited tracer uptake of lactate, pyruvate and acetate. It is concluded that a stereospecific lactate transporter exhibiting an affinity for other substituted monocarboxylic acids is operative in the sarcolemmal plasma membrane of the rabbit myocardium.