Neospora caninum and Toxoplasma gondii are closely related intracellular protozoan parasites associated with abortion in mammals. N. caninum was identified in 1984, described as a new species in 1988 and often previously misidentified as T. gondii. In this study molecular techniques were used to investigate the presence of N. caninum and T. gondii in naturally infected populations. To facilitate this a nested PCR technique, targeting the Nc5 region of N. caninum, was developed. Using this and an established T. gondii specific PCR the prevalence of N. caninum and co-infection with T. gondii were investigated in naturally infected mammals. The fox has been identified as a potential definitive host of N. caninum. PCR analysis of DNA extracted from tongue tissue determined a low prevalence of 1.4% (1/70) in British foxes. None of these foxes had detectable T. gondii DNA. Small mammals are a potential source of N. caninum infection. N. caninum was present in the brain tissues of all species investigated: Mus domesticus 3.1% (4/130), Rattus norvegicus 4.4% (2/45), Apodemus sylvaticus 3.4% (4/118) and Oryctolagus cuniculus 10.5% (6/57). T. gondii prevalences were significantly higher in all species tested. Co-infection was observed in all species except Rattus norvegicus. Investigation of vertical transmission of T. gondii in wild Mus domesticus, determined it was a common occurrence with 74.6% of foetuses from infected dams having detectable parasite DNA. A relatively high frequency of infection with N. caninum was detected in brain tissue of aborted lambs (18.9%). No significant relationship was found between N. caninum and T. gondii infection. Detection of N. caninum DNA was restricted to the brain in aborted lambs but was more widely distributed in rabbits. T. gondii DNA was detected in multiple tissues of both host species. These studies demonstrate that N. caninum is widely distributed in natural populations of mammals.