To investigate the mechanism for initiation of peroxisomal beta-oxidation by high-fat diets the time-courses of peroxisomal beta-oxidation and microsomal omega-oxidation stimulated by 20% (w/w) partially hydrogenated fish oil were studied. The relative stimulation of these two activities developed in a very similar way. We also observed an elevated level of long-chain acyl-CoA with partially hydrogenated fish oil, but not of free fatty acids. There was, however, a significant shift in the composition of free fatty acids to a higher amount of monoenes and lower amounts of 18:2 and 20:4 fatty acids. In peroxisomes purified by Nycodenz gradient centrifugation there was no lauric acid hydroxylation. This study indicates that with partially hydrogenated fish oil we obtain a parallel stimulation of reactions in two different cellular compartments. Dicarboxylic fatty acids, which are products of the omega-oxidation, had only a slight stimulatory effect on peroxisomal beta-oxidation. Therefore, the primary stimulatory agent of peroxisomal beta-oxidation and microsomal omega-oxidation is still unknown. It was speculated that this agent may activate a gene-locus responsible for both reactions.