1. Thrombin addition to human platelets stimulates L(+)lactate formation and S-D-lactoylglutathione (SDL) accumulation. 2. Monoiodoacetamide decreases lactate formation and potentiates SDL accumulation through a significant increase of dihydroxyacetone phosphate and fructose1,6bisphosphate intracellular levels both in resting and in activated platelets. 3. A similar effect is produced by exogenous methylglyoxal on L(+)lactate formation and SDL accumulation. 4. Resting platelets completely transform (1 hr at 37 degrees C) the ketoaldehyde into D(-)lactate: 5. When platelets are incubated in the presence of thrombin only 60% of the ketoaldehyde is found as D(-)lactate and the accumulated S-D-lactoylglutathione represents about the 0.7% of the initial substrate. 6. During platelet stimulation with thrombin the hemithioacetal adduct, formed as a by-product of glycolytic pathway, can be rapidly removed for important steps of cellular activation.