DNA fingerprinting has been used extensively to characterize populations of Mycosphaerella graminicola, the Septoria tritici blotch pathogen of wheat. The highly polymorphic DNA fingerprints of Mycosphaerella graminicola were assumed to reflect the action of transposable elements. However, there was no direct evidence to support that conclusion. To test the transposable element hypothesis, the DNA fingerprint probe pSTL70 was sequenced, along with three other clones from a subgenomic library that hybridized with pSTL70. Analysis of these sequences revealed that pSTL70 contains the 3′ end of a reverse transcriptase sequence plus 29- and 79-bp direct repeats. These are characteristics of transposable elements identified in other organisms. Southern analyses indicated that either the direct-repeat or reverse-transcriptase sequences by themselves essentially duplicated the original DNA fingerprint pattern, but other portions of pSTL70 contained single-copy DNA. Analysis of 60 progeny from a sexual cross between two Dutch isolates of Mycosphaerella graminicola identified several new bands that were not present in the parents. Thus, the putative transposable element probably is active during meiosis. Tests of single-spore isolates revealed gains or losses of one or more DNA fingerprint bands in 4 out of 10 asexual lines derived from isolate IPO94269. Therefore, DNA fingerprint patterns produced by the putative transposable element were capable of changes during asexual reproduction of this isolate. Probe pSTL70 did not hybridize at high stringency to genomic DNAs from other fungi related to Septoria and Mycosphaerella. These results indicate that the DNA fingerprint probe pSTL70 most likely identifies a transposable element in Mycosphaerella graminicola that may have been acquired recently, and appears to be active during both sexual and asexual reproduction.