Knowledge of intrachromosomal transpositions has until now been primarily cytological and has been limited to Drosophila and to humans, in both of which segmental shifts can be recognized by altered banding patterns. There has been little genetic information. In this study, we describe the genetic and cytogenetic properties of a transposition in Neurospora crassa. In Tp(IR-->IL)T54M94, a 20 map unit segment of linkage group I has been excised from its normal position and inserted near the centromere in the opposite arm, in inverted order. In crosses heterozygous for the transposition, about one-fifth of surviving progeny are duplications carrying the transposed segment in both positions. These result from crossing over in the interstitial region. There is no corresponding class of progeny duplicated for the interstitial segment. The duplication strains are barren in test crosses. A complementary deficiency class is represented by unpigmented, inviable ascospores. Extent of the duplication was determined by duplication-coverage tests. Orientation of the transposed segment was determined using Tp x Tp crosses heterozygous for markers inside and outside the transposed segment, and position of the insertion relative to the centromere was established using quasi-ordered half-tetrads from crosses x Spore killer. Quelling was observed in the primary transformants that were used to introduce a critical marker into the transposed segment by repeat-induced point mutation (RIP).