The specificity of random primer R143 for Aspergillus fumigatus DNA was determined in order to test its usefulness in establishing the presence of A. fumigatus DNA in fungal cultures. When PCR reaction products of these cultures were compared with those of 21 other bacterial and fungal DNA samples, R143 proved to produce a 1346 bp band with only A. fumigatus. This band has been sequenced completely and the EcoRI restriction site was used for subsequent confirmation of PCR products. The specificity for A. fumigatus DNA was also confirmed by Southern blotting. Comparison of morphological typing of Aspergillus species in cultures with PCR using R143 on DNA isolated from these cultures showed concordance in 22 of 24 cases. In two cases there was discordance: both times PCR results showed correctly the presence of A. fumigatus, initially not detected by culture. R143 is an A. fumigatus specific random primer, with potential for use in detection of A. fumigatus DNA in clinical specimens.