We previously described the construction and characterization of a Chlamydomonas genomic library in yeast artificial chromosomes (YACs). Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on the uni chromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI. Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads. This promises to make chromosomal walking in Chlamydomonas a relatively efficient enterprise. We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA. The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most any Chlamydomonas gene.