Abstract Context Insulin resistance could increase insulin requirements in type 1 diabetes (T1D). Current insulin immunoassays do not detect insulin analogs. Kinase insulin receptor (IR) activation (KIRA) bioassays specific for human IR isoforms A (IR-A) and B (IR-B) permit assessment of all circulating insulin bioactivity. We studied whether IR-A and IR-B KIRA assays are related to direct measures of insulin sensitivity or insulin doses in T1D. Design We evaluated 31 adult patients with T1D (age 45.7 ± 1.6 years, body mass index 28.8 ± 0.7 kg/m2). Serum IR-A and IR-B bioactivities were measured by KIRA bioassays. Insulin sensitivity of glucose production (Ra) was measured by the euglycemic hyperinsulinemic clamp technique in which a low insulin dose (0.4 mU/kg/min for 240 minutes) was combined with D-[3-3H] glucose infusion to measure rates of Ra and utilization and insulin action on antilipolysis from suppression of serum free fatty acids. Results Baseline circulating IR-A bioactivity was 53 ± 7 pmol/L, and IR-B bioactivity was 81 ± 11 pmol/L. Compared with baseline, insulin infusion significantly increased IR-A (P < 0.001) and IR-B (P < 0.001) bioactivities. Fasting IR-A and IR-B bioactivities were positively related to endogenous Ra (r = 0.44, P = 0.01 and r = 0.38, P < 0.05). Fasting IR-A (r = 0.43, P = 0.02) and IR-B (r = 0.47, P = 0.01) bioactivities were significantly correlated with insulin requirements and glycosylated hemoglobin (IR-A: r = 0.52, P = 0.002; IR-B: r = 0.48, P = 0.006). Conclusions Circulating IR-A and IR-B bioactivities are associated with insulin resistance, high insulin requirements, and poor glycemic control in T1D. Measurement of IR bioactivity by KIRA assays provides a tool to assess the amount of biologically active insulin in groups of T1D patients treated with insulin analogs.