Members of the genus Pediococcus have been known as brewing contaminants for more than 200 years. Traditionally, speciation of Pediococcus isolates has relied on substrate fermentation patterns. However, such patterns are variable, making species assignment difficult. Using DNA sequences from the 16S rRNA gene, the 16S-23S rRNA interspacer region, and the heat-shock 60 protein gene, seven phylogenetic units (i.e., species), including a completely new unit, for the genus Pediococcus were recently obtained by our group. The goal of the present study was to determine whether immunoblotting would parallel our phylogenetic speciation and, as such, establish if immunoblotting can be used as a phenotypic method for speciating Pediococcus isolates. Thirty-one Pediococcus isolates mostly of brewing origin were analyzed by immunoblotting after the bacterial polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transfered to polyvinylidene difluoride membranes. Twelve polyclonal antisera generated in mice or rabbits against various Lactobacillus and Pediococcus isolates were used. Comprehensive immunoblot patterns for all 31 isolates were obtained with two of the antisera, while survey immunoblot patterns for one isolate from each of the seven phylogenetic units were obtained with the remaining ten antisera. Analysis of the two sets of comprehensive blot profiles revealed in each case the same groupings of the 31 isolates as obtained in our earlier phylogenetic analyses. Visual inspection of the immunoblots revealed readily identifiable differences for isolates in each of the seven phylogenetic units; in contrast, isolates within each unit had remarkably similar blot profiles. With each of the ten survey immunoblots, the representative member of each of the seven phylogenetic units had a unique blot pattern. Furthermore, each of the 12 antisera gave a unique banding pattern for each unit. The immunoblotting profiles thus were found to depend on the bacterial isolate (or, more specifically, the phylogenetic unit of the isolate) and the polyclonal antisera used. Since the immunoblot profiles obtained with different antisera gave isolate groups fitting with those determined phylogenetically, we believe that immunoblotting is a valid and readily deployed phenotypic method for speciation of Pediococcus isolates.