Polymerase chain reaction (PCR) analysis is a widely used method for detection of Borrelia burgdorferi DNA in biological specimens, including ticks. Studies have demonstrated that substances present in mammalian blood can inhibit PCR amplification. This would limit the utility of PCR for determination of B. burgdorferi infection in engorged ticks that have taken a blood meal from a human or other animal host. To systematically assess the potential for such inhibition, nymphal Ixodes scapularis, which had acquired B. burgdorferi as larvae, were fed on rats. These engorged ticks were lysed in standard PCR lysis buffer and aliquots were subjected to PCR analysis; 0 of 56 were PCR positive. An equivalent cohort of unfed (unengorged) ticks had an infection rate of 19% (11 of 57) as determined by identical PCR analysis (P = 0.0006, by Fisher’s exact test). When lysates from the engorged ticks were spiked with the 500 prelysed B. burgdorferi, none of the samples yielded a positive PCR signal, indicating the presence of inhibitory substances. Consistent with this observation, PCR amplification of the original engorged tick lysates after extraction with a DNA extraction kit, resulted in detection of B. burgdorferi DNA in 13 specimens (23%). Furthermore, when 500 prelysed B. burgdorferi were added to the treated extracts, all samples (56 of 56) were PCR positive. Thus, extraction resulted in removal of inhibitors of PCR amplification present in unprocessed engorged tick lysates. Furthermore, additional titration experiments showed that some inhibitory substances may also be present in unfed ticks, although this inhibition does not completely prevent detection of B. burgdorferi DNA in unprocessed lysates. This study clearly demonstrates that inhibitors of PCR amplification are present in engorged ticks and prevent accurate determination of B. burgdorferi infection rates by this method unless steps are taken to remove such inhibitors.