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Summary. When guinea-pig spermatozoa were suspended in a minimal culture medium (MCM-PL), 2-deoxy-d-glucose (100 μm) and 2-amino-2-deoxy-d-glucose (100 μm) were potent inhibitors of the acrosome reaction without affecting the sperm motility, whereas the N-acetyl derivative 2-acetamido-2-deoxy-d-glucose (2 mm) had no inhibitory effect. The addition of d-glucose (2 mm) partly inhibited the percentage acrosome reaction of spermatozoa suspended in Medium MCM-PL, but dl-aglycerophosphate (2 mm) and myo-inositol (2 mm) had no effect. In addition, dl-α-glycerophosphate (2 mm) did not overcome the inhibitory effect of 2-deoxy-d-glucose on the sperm acrosome reaction. The inhibitory action of 2-deoxy-d-glucose (100 μm) on the sperm acrosome reaction assessed after a 3-h incubation was irreversible and was only completely effective if the sugar was added within 30 min of the start of incubation. When spermatozoa suspended in Medium MCM-PL were treated with 2-deoxy-d-glucose (100–200 μm) for an extended incubation up to 6 h, the inhibitory effect of 2-deoxy-d-glucose was partly overcome. Spermatozoa treated with 2-deoxy-d-glucose had significantly reduced concentrations of ATP after incubation for 2 h in Ca²⁺ -free media, compared with the ATP concentrations of spermatozoa preincubated for 2 h in Ca²⁺ -free media that supported acrosome reactions. The addition of Ca²⁺ (5 mm) caused a rapid decrease in ATP concentrations of spermatozoa suspended in Medium MCM-PL, while the addition of the monovalent ionophore monensin (50 μm) and Ca²⁺ stimulated sperm acrosome reactions as well as an additional decline in the sperm ATP concentrations. However, monensin (50 μm) in the absence of Ca^{2 +} caused only a slight decline in the sperm ATP concentrations over the 15-min incubation period. The depletion of the sperm ATP concentrations by 2-deoxy-d-glucose may retard completion of the capacitation process and the resultant acrosome reaction.