Dog sera (n = 118) were tested for antibodies recognizing Borrelia (B.) burgdorferi sensu stricto strain B31 (ATCC 35210) antigens. In total, 18 of the dog sera gave positive results in a whole cell sonicate ELISA (WCS ELISA). These positive sera were further evaluated by immunoblot assay, utilizing a whole bacterial lysate as antigens. 94.4% (17 of 18) of the dog sera reacted with immunodominant antigens at 20-22 kDa (protein C, pC), 31 kDa (outer surface protein A, OspA), 34 kDa (outer surface protein B, OspB), 41 kDa (flagellin), 60 kDa ("common antigen"), and/or 100 kDa (presumably p100). Sera recognizing pC (20-22 kDa) and antigens > 94 kDa always detected the highest number of antigen bands, indicating the specificity of those antigens in serological diagnosis. The results clearly demonstrate that the WCS ELISA is a useful tool for testing sera of dogs for antibodies against B. burgdorferi. However, positive results should be confirmed by immunoblot, using WCS as antigen. According to the presented data, we recommend criteria for B. burgdorferi immunoblots using dog sera as follows: sera have to be considered as positive if they detect the 41 kDa flagellin, and two of the 5 immunodominant antigens, namely > 94 kDa (presumably p100), 60 kDa ("common antigen"), 34 kDa and 29-31 kDa (OspB and OspA, respectively) and 20-22 kDa (pC). If sera only recognize the 41 kDa flagellin, this result is equivocal, requiring testing a second serum sample 4 to 8 weeks later.