With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.