We isolated the LYS2 gene of S. cerevisiae on an autonomously replicating plasmid in a four-step procedure. First, we identified a recombinant plasmid which expressed a lys2 complementing activity upon yeast transformation of a lys2 mutant. Second, we determined the boundaries of the corresponding transcribed sequence in this plasmid by S1 nuclease mapping of the mRNA. Third, we inactivated the functional chromosomal copy coding for the lys2 complementing activity by directed integration of a plasmid that carried an internal fragment of the transcribed sequence. Fourth, we showed by a complementation test with an authentic lys2 mutant that the integration had inactivated the chromosomal LYS2 gene. This proved that the plasmid isolated in the first step indeed carried the LYS2 gene and not a suppressor of the lys2 mutation. The gene is unusually long (4.0 kb). It was used as hybridization probe in comparing LYS2 regions in various yeast strains and in a first construction of LYS2 based yeast vectors. Such vectors could be very useful because of the easy selection of lys2 mutants from any S. cerevisiae strain.