AbstractA molecular understanding of peroxisome biogenesis depends upon the analysis of peroxisomal proteins. Here we describe the isolation of the 3‐oxoacyl‐CoA thiolase of the peroxisomal β‐oxidation system from Saccharomyces cerevisiae as a dimer of identical subunits, each with a molecular mass of 45 kDa. Monospecific polyclonal antibodies were raised against the purified enzyme, and its peroxisomal origin was demonstrated by immunoblotting of subcellular fractions as well as by immunogold labelling. We also show that these antibodies could be suitable for an immunofluorescence microscopy screening of yeast mutants affected in peroxisome assembly.