[U-14C]Sucrose and D-[1-14C]mannitol were used to determine the tritiated water space of human spermatozoa to validate these compounds as markers for the extracellular space. Calculations based on 0.03 mM-[U-14C]sucrose gave a negative water space. The water space estimated with 0.03 mM-D[1-14C]mannitol was unstable but a stable result was obtained with 0.3 mM-D-[1-14C]mannitol in incubations up to 2 h. The mean water space was 2.21 +/- 0.106 microliters/10(8) spermatozoa (mean +/- s.e.m. for 6 batches of pooled semen). The water space was decreased or abolished by Triton X-100, cold shock, sonication or hypotonic treatment. The water space responded to changes in the osmolarity of the medium by increasing in dilute media. It is concluded that mannitol is an effective extracellular marker for human spermatozoa if concentrations greater than or equal to 0.3 mM are used. When the kinetics of the uptake of 2-deoxyglucose by the spermatozoa were studied by using mannitol as an extracellular marker, the transport was saturable and was inhibited by cytochalasin B. The Km was 1.6 +/- 0.33 mM and the Vmax was 4.2 +/- 0.52 nmol/10(8) spermatozoa/10 sec (mean +/- s.e.m., n = 4).