Twenty five strains of Yersinia enterocolitica serogroup O3, were isolated from human enteritis and studied for heat-stable enterotoxin production. Enterotoxin production was found even in the crude supernatant fluid of cultures that had been stored in stock agar for a year. According to the suckling mice and rabbit gut loop tests, after 1 to 5 years storage the filtrate showed heat-stable enterotoxin activity only in a purified and concentrated form. Following more than 5 years storage positive results could be obtained only in rabbit gut loop test. After 9 years the freeze dried strains still showed a full capacity of heat-stable enterotoxin production. Studies with concentrated substances showed that even after more than 9 years, there was no spontaneous loss of heat-stable enterotoxin production, only quantitative changes occurred. The methanol solubility of the heat-stable enterotoxin of Y. enterocolitica is--as distinct from the heat-stable enterotoxin of Escherichia coli--homogeneous and only the methanol soluble fractions showed any activity. The activity of methanol soluble enterotoxin from several years old subcultures could be demonstrated in an isolated rabbit gut loop model even when it failed to show any activity in suckling mice.