We have constructed and characterized a Chlamydomonas reinhardtii total genomic library in yeast artificial chromosomes (YACs). The library contains 7500 clones with inserts ranging in size from 100-200 kb. The representation of the library was assessed by screening one-third of it with a probe derived from the dispersed repeat, Gulliver, which occurs approximately 13 times in the genome. At least 10 of these Gulliver loci were isolated within 15 independent YACs. Two of these YACs encompass the Gulliver element designated G, which was reported to map to the uni linkage group (ULG). The end clones of these two YACs have been genetically mapped by RFLP analysis in an interspecific cross and thereby shown to be closely linked to the APM locus on the ULG. A third uni-specific YAC has also been isolated and its ends have been mapped by RFLP analysis. Genetic and RFLP analysis of these and other YACs indicates that the frequency of chimeric YACs in the library is very low. The library was constructed in a second generation vector that enables plasmid rescue of YAC end clones as well as copy number amplification of artificial chromosomes. We provide evidence that amplification of intact YACs requires a rad1:rad52 yeast strain.