We developed a method that allows the measurement of muscle lactate transport in humans. The transport studies were carried out with giant (1.8- to 36-microns-diam) sarcolemmal vesicles obtained by collagenase treatment of needle biopsy material. Marker enzyme analyses demonstrated that the vesicular membrane is predominantly of sarcolemmal origin, contamination with sarcoplasmic reticulum membranes is very low, and mitochondrial membranes are not a major contaminant. The vesicles were loaded with labeled lactate, and the efflux was measured. The system displayed saturation kinetics and inhibitor sensitivity. In equilibrium exchange experiments (pH 7.4, 21 degrees C), the Michaelis-Menten constant (Km) for the carrier-mediated flux was 30 +/- 8 (SD) mM and maximal transport rate (Vmax) was 184 +/- 24 pmol.cm-2.s-1 (142 nmol.mg protein-1.min-1). In zero-trans efflux experiments, Km was 24 +/- 8 mM and Vmax was 81 +/- 11 pmol.cm-2.s-1 (63 nmol.mg protein-1.min-1). In infinite-cis experiments with a variable lactate concentration on the outside of the vesicles, Km was 8 +/- 4 mM and Vmax was 136 +/- 9 pmol.cm-2.s-1 (105 nmol.mg protein-1.min-1). Thus, the system displayed transacceleration. Low pH (6.4) had no significant effect on equilibrium exchange experiments, whereas in zero-trans experiments low pH at the trans side inhibited the flux by 50%. We concluded that lactate transport can be studied in giant vesicles obtained from a single human muscle biopsy. Our data provide evidence for the existence of a lactate carrier in human sarcolemma. This transport system must be taken into account in models of human lactate kinetics.