OBJECTIVE: To test the ability of a nested PCR assay to detect Eimeria macusaniensis at various stages of infection in alpacas. ANIMALS: 4 healthy adult alpacas with no detectable E. macusaniensis. PROCEDURES: Alpacas were inoculated with 2 × 10(4) sporulated oocysts. Serial fecal samples collected during the next 38 days were tested via sucrose flotation and PCR assay. RESULTS: Oocyst passage was detected via fecal flotation in all 4 alpacas 31 to 35 days after inoculation. Three had positive results for PCR assays on samples obtained 7 to 14 days after inoculation. One alpaca subsequently was removed from the study because of weight loss and inappetence. Two remaining alpacas had positive PCR reactions 28 and 31 days after inoculation, up to 7 days before oocysts appeared in the feces. All fecal samples with positive results for flotation also had positive results for PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: The PCR assay was able to detect early (7 to 14 days) and late (28 to 31 days) prepatent infection. These positive results suggested that the assay could have been detecting DNA unassociated with oocysts or detecting shedding earlier than has been previously recognized. The gap between the early and late detection periods may not be evident in alpacas receiving a larger or continuous inoculum, as might occur with natural infection. Use of a PCR assay for analysis of fecal samples may be valuable for detection of E. macusaniensis during the prepatent period, thus aiding in the identification and control of infected animals.