BACKGROUND: In this work, a comprehensive study of the respirometric and physical-chemical methods has been performed to evaluate the information provided by these two methodologies in order to assess the biodegradable organic matter fractions of wastewater. RESULTS: First, an analysis was performed of the influence of the initial substrate on biomass ratio (F-0/X-0) in the assessment of readily biodegradable organic matter, S-S, through respirometric experiments. In order to achieve an adequate assessment of the S-S component, similar conditions (given by the initial F-0/X-0 ratio) must be employed in experiments that are carried to determine S-S and Y-H (heterotrophic yield). Second, a comparative analysis of respirometric and physical-chemical methods was carried out. The respirometric analysis yielded a lower value of the S-S component than the one using the physical-chemical method. Moreover, the respirometric analysis cannot measure the total content of slowly biodegradable organic matter, X-S, since this method is only capable of determining the readily hydrolyzed biodegradable substrate. CONCLUSIONS: Considering the results, an adequate characterization method of the S-S component has been proposed. This method provides readily and slowly hydrolyzable biodegradable organic matter fractions. (C) 2010 Society of Chemical Industry