Sera from vaccinated lambs with Brucella melitensis Rev.1 and naturally infected sheep with Brucella melitensis biovar 3 as well as Brucella free sheep, were examined using standard serological tests. No great difference in standard serological tests results between vaccinated and infected animals. These sera were also analyzed for their serological reactivity against whole killed cell (WKC) and cytosoluble protein extract (CPE) antigens using ELISA. The present study showed that ELISA with WKC was unable to differentiate infected sheep from those vaccinated with Rev.1 while ELISA with cytosoluble proteins antigen may able to differentiate antibody response of infected animals from vaccinated ones. Using immunoblot technique, sera from naturally infected animals showed strong antibody reactivity to 28.48KDa and variable reactions to 58.80, 49.70, 39.60, 32.47 and 18.00 KDa. However sera from Rev.1 vaccinated animals showed less intense antibody reactivity which only observed against proteins with molecular masses of 49.70 and 39.60 KDa. It is likely that cytosoluble proteins may provide useful serological reagent for differentiation be- tween infected and vaccinated sheep.