Electrophoretic karyotype of yeast strain A364a was obtained by pulsed field gel electrophoresis and the position of chromosome V on such karyotype was determined by means of dot hybridization with chromosome V-specific probe URA3. By cloning partially digested BamHI fragments of this chromosome DNA into integrative vector Yip5, a gene library specific to this chromosome was constructed. The number of the recombinants was much more than theoretically required. After screening probe-homologous fragments from this library and analysing such fragments with restriction enzymes BamHI, EcoRI, HindIII, PstI, and SalI, a fine physical map covering about 9.4% of A364a chromosome V (which was estimated as 620kb) was constructed. Further colony hybridization with boundary clones will enable us to "walk" throughout the whole chromosome.