Protein Z was recently shown to act as an essential cofactor for protein Z-dependent protease inhibitor, a potent downregulator of coagulation Factor Xa. Thus, deficiency of protein Z is hypothesized to lead to a prothrombotic state, but two publications reported opposing results for the relationship of protein Z levels with ischemic stroke in young European subjects (mean age 33-40 years). We performed a study of stroke in a different ethnic population of greater mean age (57 years) to further clarify this issue. An ELISA was developed to measure protein Z antigen in 154 patients with ischemic stroke and in 206 controls in a largely Hispanic population. Low plasma protein Z values were significantly associated with ischemic stroke except in diabetic subjects and females. The mean protein Z value was significantly lower in stroke cases than in controls for nondiabetic subjects [1.78 +/- 0.77 (S.D.) versus 2.28 +/- 0.88 microg/ml, P < 0.0001] and for males (1.90 +/- 0.90 versus 2.42 +/- 0.99 microg/ml, P = 0.0004). Stroke risk was higher in subjects with protein Z levels at or below the fifteenth percentile of controls (=1.46 microg/ml). The odds ratios were: 2.6 for all subjects (95% C.I. = 1.5-4.3); 3.8 for nondiabetic subjects (95% C.I. = 2.2-6.8); and 3.6 for males (95% C.I. = 2.0-6.4). This study also revealed high protein Z levels associated with high triglyceride levels in controls (P = 0.015). Protein Z is suggested to be a physiologic downregulator of blood coagulation and low protein Z levels are associated with increased risk of ischemic stroke, particularly in males and in the absence of diabetes.