Improvements of the activity staining method for yeast alcohol dehydrogenases (ADH) and measurements of the relative mobility (Rm) and the staining activity of ADH isozymes were investigated. Cell-free extracts of yeasts were submitted to the poly-acrylamide gel electrophoresis and the ADH isozymes in the gel were activity-stained by a modified method of activity staining of SCHWARTZ and ENDO with measurements of Rm's of ADH isozymes taking the Rm of xylene cyanol FF as 1.00. In most cases, the ADH isozyme pattern in the gel of Shochu yeasts grown in shaking cultivation was different from that of the statically grown cells. Almost the same results of Rm's and patterns of ADH isozymes were obtained under the same experimental conditions. Measurements of the staining activity of ADH isozymes in gels were carried out with a chromatographic scanner. The relation between the staining activity Y (staining units, su) of the ADH (Oriental Yeast, Co., Ltd.) and the amount of enzyme protein X ( u g) followed the equation: Y=8.36×10³X-1.21×10³ (2.00×10³5. Ca²⁺: 1mm). Influences of various staining conditions divalent cation concentrations, pH and substrate alcohols in the staining solution on the staining activities of ADH isozymes were investigated. An increasing staining activity was observed by 10-30% for the Shochu yeasts by the addition of CaC1₂ or MgCl₂ to the staining solution to give a concentration of 0.5 to 1.0mm. Relations between the staining activity of ADH and the fermentation rate, or alcohol production rate were also checked. The above results indicated that ADH isozymes were characterized with the Rm's and the substrate specificity in the activity-stained polyacrylamide gel, and that changes in the genetic background of yeasts would be detected by the Rm's and the substrate specificity, and by means of measurements of the staining activities of ADH isozymes.