We have positioned amplified fragment-length polymorphism (AFLP) markers directly on the genome sequence of a complex organism, Arabidopsis, by combining gel-based AFLP analysis with in silico restriction fragment analysis using the published genome sequence. For placement of the markers, we used information on restriction fragment size, four selective nucleotides, and the rough genetic position of the markers as deduced from the analysis of a limited number of Columbia (Col)/Landsberg (Ler) recombinant inbred lines. This approach allows for exact physical positioning of markers as opposed to the statistical localization resulting from traditional genetic mapping procedures. In addition, it is fast because no extensive segregation analysis is needed. In principle, the method can be applied to all organisms for which a complete or nearly complete genome sequence is available. We have located 1,267 AFLP Col/Ler markers resulting from 256 SacI+2, MseI + 2 primer combinations to a physical position on the Arabidopsis genome. The positioning was verified by sequence analysis of 70 markers and by segregation analysis of two leaf-form mutants. Approximately 50% of the mapped Col/Ler AFLP markers can be used for segregation analysis in Col/C24, Col/Wassilewskija, or Col/Cape Verde Islands crosses. We present data on one such cross: the localization of a viviparous-like mutant segregating in a Col/C24 cross.