Differences in bioavailability of hydrophobic organic compounds (HOC) to benthic deposit feeders have been related to differences in sediment-HOC contact time and sequestration (formation of slowly desorbing fractions) status. As a consequence, it was postulated that contact time and/or sequestration should be incorporated into risk assessment. In the present study, we investigated the effect of contact time on the bioavailability and sequestration of different classes of HOC. For this purpose, we simultaneously measured the steady-state accumulation into benthic oligochaetes (Tubificidae) and the distribution over rapidly and slowly desorbing fractions in laboratory-contaminated sediment at different contact times. The decrease in rapidly desorbing fractions (Frap) of polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and 2,2-bis (4-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE) after a contact time of 959 d did not exceed a factor of 1.2. Similarly, the reduction in bioavailability was a factor of 2.3 at maximum, indicating that long contact times do not necessarily result in pronounced bioavailability reduction. For chlorobenzenes, the bioavailability was reduced with a factor of 5 to 18. This decrease corresponded with a pronounced reduction in Frap, which was attributed to losses of rapidly desorbing compounds. Over 75% of the variation in biota-to-sediment accumulation factors (BSAFs) of the PAHs and chlorobenzenes at the three contact times could be explained by differences in Frap. The present study provides evidence of a relationship between sequestration status and bioavailability of HOC to benthic deposit feeders.