Canine systemic lupus erythematosus (SLE) has a similar disease expression as human SLE, but the serological characterisation of the canine disease is as yet incomplete. In the present study, we examined the specificity of antinuclear antibodies (ANA) in indirect immunofluorescence (IIF) positive canine sera. Sixty-four canine IIF ANA positive sera were characterised using HeLa cell nuclear extract immunoblots and recombinant U1-70K ELISA. We compared these results with a previously shown concordance between indirect immunofluorescence and immunodiffusion in canine SLE serological diagnosis. One canine serum reacting with Sm proteins was observed, and five canine sera presented anti-RNP autoantibodies against the antigens 70K, A, C, and/or B/B'. The autoantigen most frequently recognised was a 43 kDa nuclear protein, previously described as hnRNP G. This prominent canine autoantigen was missing in the commercially available extract designed for immunodiffusion testing of human sera. Other prominent canine autoantigens were found not to be identical with the principal human ones, thus making present human test systems deficient for the use in canine systemic connective disease diagnosis. The development of antigenic extract designed for canine autoimmune autoantigens is necessary in order to make immunodiffusion a useful method in canine diagnosis. The anti-RNP positive canine sera were examined in more detail and we found that the human major antigenic region of the most prominent RNP antigen, the U1-70K protein, also is targeted by canine autoantibodies. Thus, the response against the RNP antigen seems to be conserved between man and dog.