Multilocular echinococcosis caused by the larval stage of Echinococcus multilocularis is an important zoonosis with high mortality. In Hokkaido, Japan, although the prevalence among inhabitants has been very low, the areas infested with the parasite are rapidly spreading in recent years. Therefore, we improved on the procedure of the enzyme-linked immunosorbent assay (micro-ELISA) for serological diagnosis of multilocular echinococcosis to carry out the epidemiological survey. A micro-ELISA plate (M129A, DYNATECH), antigen of E. multilocularis cyst extract, peroxidase-conjugated anti-human IgG (MILES-YEDA), O-phenylenediamine and a plate reader (Multiskan MC, TITERTEK) were used for the assay. Reactions were performed with 0.1 ml of reagents in wells of plate at each step and 0.1 ml of 0.2N H2SO4 was finally added for stop of the reaction. The optimum condition was determined by block titration, as 10 micrograms/ml of protein for antigen concentration, 1:40 for serum dilution and 1:4,000 for conjugate dilution. In this assay system, 30 serum samples could be examined in one plate. After correcting the absorbance of negative control to 0.300, positive range was defined as more than 0.600 which was twice the value of negative control. Doubtful positive range was defined as between 0.450 and 0.600, and negative range as less than 0.450. According to this criterion, 30 serum samples of proven patients with multilocular echinococcosis were examined. Positive and doubtful positive reactions were found from 20 sera (66.7%) and 7 sera (23.3%), respectively. Three patient sera (10.0%) showed negative reactions.(ABSTRACT TRUNCATED AT 250 WORDS)