Liquid culture filtrates (CF) of Mycobacterium avium subsp paratuberculosis (Map) contain more antigenic proteins that react with sera from infected cattle. The aim of this study was to identify proteins with potential diagnostic value. CF MAP proteins were separated by SDS-PAGE one-dimensional electrophoresis technology. The CF proteins were harvested from supernatants of liquid cultures in stationary-phase and concentrated by size exclusion filtration. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa). The antigenicity of CF proteins was determined by 1-DE immunoblotting with sera of four different cows naturally infected with MAP. The sera reacted strongly with proteins in the range of 20 - 40 kDa. When sera from different infected cattle were tested by immunoblotting with CF proteins, a high degree of variability in protein binding patterns was observed. Additionally, when bovine sera were absorbed with environmental mycobacteria, namely M. avium, M. phlei, M. terrae, M. scrofulaceum and M. smegmatis, and tested by immunoblotting, there were no major differences in the antigenic patterns between absorbed and non-absorbed sera. These results indicate that serological tests like ELISA for bovine paratuberculosis may be improved by using CF proteins, and the serum preabsorption step using environmental mycobacteria could potentially be eliminated from the ELISA procedure.