All Leptosphaeria maculans field isolates displayed a minichromosome (MC) clearly separated from the overall electrokaryotype following pulsed-field gel electrophoresis. MCs exhibited a length polymorphism ranging from 650 to 950 kb. Tetrad analyses revealed the parental inheritance of MC length polymorphism (50% of the tetrads) or else the generation of novel-sized MCs (27%), which suggested that recombination occurred between MCs. Nineteen percent of the tetrads displayed a lack of the MC band in the electrokaryotype for one or two of the four resulting genotypes. Crosses between isolates carrying or lacking MCs revealed non-Mendelian segregation and suggested that some isolates could display at least two copies of the MC. Only repeated sequences hybridising to all chromosomes were isolated from the MC. Finally, saprophytic or parasitic fitness was not modified when isolates apparently lacked the MC. All these data suggested that the L. maculans MC behaves like a 'B' chromosome.