To detect oocysts of Neospora caninum in dog feces and to determine the excretion pattern in dogs from specialized dairy farms in Costa Rica, a total of 265 fecal samples from 34 dogs were collected at intervals from February to August 2005. Fecal samples were examined for N. caninum-like oocysts microscopically, by DNA detection using the polymerase chain reaction (PCR), and by bioassay. N. caninum DNA was detected by PCR in four fecal samples, twice from one dog, but oocysts were not detected microscopically in these dogs. Sera of 31 of 34 dogs were tested for antibodies to N. caninum by a competitive-inhibition ELISA (VMRD®). Fifteen (48.4%) of 31 dogs had antibodies to N. caninum by ELISA. Seroconversion was not found in 28 dogs that were bled twice, 4 months apart (March and July 2005). Only one dog tested positive to N. caninum by both ELISA and PCR. This is the first report of finding N. caninum DNA in feces of naturally infected dogs in Costa Rican dairy farms.