The interdomain acidic region a1 is a unique structural feature of coagulation factor VIII (FVIII) and may mediate the proteolytic activation of FVIII and the inactivation of FVIIIa. We report an individual with a Tyr346-->Cys substitution within region a1, who presented with a one-stage FVIII activity (FVIII:C) of 0.34 iu/ml (normal range 0.5-2.0) but normal two-stage FVIII:C and FVIII antigen values. In a factor Xa (FXa)-generation assay for FVIII in which the activation time with thrombin was varied, the variant plasma showed normal FVIII:C at both short and long activation times. However, at intermediate activation times the FXa generation of the variant plasma was less than that of normal pooled plasma. In a modified one-stage FVIII:C assay in which partially purified FVIII was activated with thrombin at low concentrations, the variant FVIII showed less activation than wild-type FVIII, although this defect corrected with increasing concentrations of thrombin. When partially purified variant FVIII was activated with a large molar excess of thrombin, the subsequent rate of decay of FVIII:C was greater for variant FVIII. The complex defects in activation and inactivation displayed by FVIII Tyr346-->Cys support the hypothesis that the a1 sequence is a key regulator of FVIII activity.