For the genetic analysis of molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, we isolated mutants of Arabidopsis thaliana that are impaired in vascular patterning. Microscopic examination of the cotyledonary venation of 3,400 M(3) lines led to the identification of 12 mutant lines. Genetic analysis of 8 of these mutant lines indicated that vein pattern formation in these lines resulted from monogenic recessive mutations in 7 different genes, designated VAN1 through VAN7. Mutations in VAN1 through VAN6 genes caused fragmentation (disconnection or partial loss) of lateral veins of the cotyledon and tertiary veins of the rosette leaf whereas they were less injurious to the formation of major veins. Detailed characterization of the van3 mutant using pAthb8::GUS and pTED3::GUS, as molecular markers for the early stage of vascular tissue formation showed that the provascular tissue of the cotyledonary lateral veins was differentiated in fragments during late embryogenesis. These phenotypes of the van mutants are discussed in relation to the auxin signal flow canalization hypothesis and the diffusion-reaction prepattern hypothesis, with the fragility of the continuity in the minor vein formation favoring the latter hypothesis.