A clinical isolate of nontoxigenic Vibrio cholerae O1 that caused intestinal fluid accumulation (FA) in adult mice produced proteolytic, hemolytic, and cytotoxic activities in in vitro assays. The linkage of these secreted factors to the FA activity was studied by transposon (TnphoA) mutagenesis. Ten of the 12 TnphoA insertion mutants that were defective for proteolytic activity produced FA, hemolytic and cytotoxic activities; the remaining two mutants lost these latter three activities. These results indicate that FA activity is independent of proteolytic activity but closely associated with cytotoxic and hemolytic activities. Our results with the adult mouse model and a nontoxigenic V. cholerae 01 are in general agreement with previous studies that demonstrated linkage of cytotoxin and hemolysin of toxigenic V. cholerae O1 and non-O1 with FA activity in rabbit ileal loops.