A three‐way crossover study in 27 human volunteers was conducted to characterize the pharmacokinetics and to assess the dose proportionality of 100 mg, 200 mg and 300 mg strengths of a novel once‐a‐day tramadol controlled‐release tablet (Tramadol Contramid^® OAD) following single‐dose administration. Serial blood samples were collected at predefined timepoints over a 48 h period and racemic tramadol and O‐desmethyltramadol concentrations in plasma were determined using a validated LC‐MS/MS method. Pharmacokinetic parameters were derived using noncompartmental methods. Following dose normalization and logarithmic transformation of concentration‐dependent parameters, the results were compared using analysis of variance (ANOVA). The residual variability thereby obtained was used to construct 90% classical confidence intervals. The two one‐sided tests procedure was used for all pairwise comparisons. Dose proportionality was concluded since the 90% CI for the ratio of geometric means was included in the acceptance range of 0.80–1.25 for all comparisons. Copyright © 2007 John Wiley & Sons, Ltd.