OBJECTIVE: To measure the contribution of bedtime intermediate-acting human insulin on the morning plasma insulin profiles after injection of the rapid-acting insulin analogs lispro and aspart in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: A total of 14 patients with type 1 diabetes, aged 35 +/- 13 years (mean +/- SD), participated in this single-blind, randomized crossover study. After taking their usual injection of human intermediate-acting insulin the night before, they were given insulin aspart or insulin lispro (10 units) before a standardized breakfast. The contribution of continuing absorption of the human insulin was measured using a monoclonal antibody not cross-reacting with insulin aspart or lispro, whereas the contribution of the analogs was estimated by subtraction after measurement of all plasma free insulin using an antibody cross-reacting equally with human insulin and both analogs. RESULTS: The correlation coefficient of the fasting free insulin concentrations measured with both insulin methods was 0.95. Fasting free insulin was 95 +/- 25 pmol/l before administration of insulin aspart, when determined with enzyme-linked immunosorbent assay detecting only human insulin, and 71 +/- 20 pmol/l before administration of insulin lispro (NS). Both insulin analogs gave marked peaks of free insulin concentrations, lispro at 40 +/- 3 min and aspart at 55 +/- 6 min after injection (P = 0.01). The later part of the profiles, from 4.5 to 5.5 h after injection, were similar and showed almost no contribution of the insulin analogs. CONCLUSIONS: The combination of insulin assays that detect human insulin only or both human insulin and analogs provides a new tool for studying insulin pharmacokinetics. Using this technique, we showed that 4.5 h after administration of the rapid-acting insulin analogs lispro and aspart, the free insulin levels are almost only attributable to the intermediate-acting insulin given at bedtime.