Comparison of the initial (3)H/(14)C ratios in specifically labelled d-glucose 6-phosphates with the final ratios in myo-inositol produced by glucose 6-phosphate-d-myo-inositol 1-phosphate cyclase from rat testis showed that, during the conversion, the hydrogen atoms at C-1 and C-3 were fully retained, one hydrogen atom was lost from C-6, and that at C-5 was apparently retained to the extent of 80-90%. The loss of (3)H could not be stimulated by addition of unlabelled NADH, and when unlabelled substrate was used (3)H from [(3)H]NADH and [(3)H]water was not incorporated. Treatment of the enzyme with charcoal abolished the activity, and this was restored to 25-50% of the original activity by NAD(+). The charcoal-treated enzyme again apparently gave 85% retention of hydrogen with [5-(3)H]glucose 6-phosphate as substrate in the presence of NAD(+) alone, but the retention was decreased to 65% with excess of NADH. The results are interpreted as indicating that the cyclization proceeds by an aldol condensation in which C-5 is oxidized by NAD(+) in a tightly-bound ternary complex, and that the apparent loss of (3)H when untreated enzyme is used is due to an isotope effect. It is suggested that after treatment with charcoal some exchange of NADH with an external pool may take place.