The levels of tissue kallikrein are reduced in humans with hypertension, cardiovascular and renal diseases. There is little information on the participation of human tissue (renal/urinary) kallikrein (hK1) in type 1 diabetes mellitus (DM), and, no information concerning the role of hK1 was reported in gestational diabetes mellitus (GDM). The present study evaluated the roles of insulin and hyperglycemia on urinary hK1 activity in type 1 DM and in GDM. Forty three type 1 DM patients (535 years, disease duration 5 years, receiving insulin, HbA1c > 7.6%) were selected. Forty three healthy individuals, paired according to gender and age, were used as controls. Thirty GDM patients (18−42 years, between the 24th and 37th weeks of pregnancy, recently diagnosed, not under insulin therapy) were also selected. Thirty healthy pregnant and thirty healthy non−pregnant women (18−42 years) were selected as controls. A random midstream urine was used. hK1 amidase activity was estimated with D-Val-Leu-Arg-Nan substrate. Albumin was determined with Coomassie Brilliant Blue reagent. Creatinine was determined by Jaffe's method. hK1 specific amidase activity was expressed as μM/(min/mg creatinine to correct for differences in urine flow rate. hK1 specific amidase activity was significantly higher in the urine of type 1 DM and GDM patients, respectively, than in controls. These findings suggest that hyperglycemia rather insulin is involved in the mechanism of increased hK1 specific amidase activity in the urine of both type 1 DM and GDM patients. Albumin/creatinine ratio was increased in type 1 DM, GDM and healthy pregnant women.