The 5.8 S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus mosseae FL156 and UK118 were amplified by polymerase chain reaction (PCR) using ITS1 and ITS4 as primers. The amplification product from template DNA of UK118 was cloned and sequenced (569 bp); the amplified DNA from FL156 was sequenced directly (582 bp). There was a 95% sequence similarity between DNAs amplified from the two isolates; in contrast, major dissimilarities with partial sequences of seven other glomalean taxa were observed. Four oligonucleotide sequences unique to Glomus mosseae were identified as potential primers. Their specificity to Glomus mosseae was assessed by PCR amplification of genomic DNA from spores from 36 glomalean fungi: 13 isolates of Glomus mosseae, two Glomus monosporum, 10 other Glomus isolates, and 11 other glomalean taxa from each of four other genera. The Glomus mosseae isolates were from a broad range of temperate zone agricultural soils. Oligonucleotide pair GMOS1 : GMOS2 primed specific amplification of an oligonucleotide sequence (approximately 400 bp) present in all Glomus mosseae isolates and two isolates of the closely related Glomus monosporum. This primer pair did not prime PCR when the template consisted of DNA from any of the other glomalean fungi or any of the nonmycorrhizal controls. In addition, a 24-mer oligonucleotide, designated GMOS5, hybridized with Glomus mosseae and Glomus monosporum DNA amplified by PCR using primer pairs ITS1 : ITS4 and GMOS1 : GMOS2. Colony-blot assays showed that GMOS5 hybridized to 100% and 97% of E. coli pUC19 clones of amplification products from Glomus mosseae FL156 and UK118 DNA templates, respectively, indicating that nearly all clones contained an homologous sequence. GMOS5 was used successfully to detect specifically Glomus mosseae in DNA extracted from colonized sudan grass (Sorghum sudanense L.) roots and amplified by PCR using the primer pair GMOS1 : GMOS2. The results confirm several previous indications that Glomus mosseae and Glomus monosporum are indistinguishable taxonomic entities.