Biological activities of the eight holotoxin preparations (the untagged and singly and doubly tagged native holotoxin controls and the variant holotoxins with zero to four wt BS and one or two tags) were initially tested in an overnight assay on mouse Y1 adrenal cell monolayers, on which cholera toxin causes an easily scorable morphological change (rounding of intoxicated cells). In this assay, approximately 2 ng of unnicked native cholera toxin caused rounding of 75 to 100% of the cells (48 U/100 ng). The G33D holotoxin showed some rounding at only the highest concentration tested (33 ng, extrapolating to less than 1 U/100 ng). All holotoxin variants with one or more BS showed rounding of Y1 cells at between 24 and 48 U/100 ng, showing that a single GM 1 binding site is sufficient to intoxicate mouse Y1 cells at near-wt levels. Failure to detect significantly less toxicity after overnight exposure of mouse Y1 adrenal cells to holotoxin variants with from 1 to 4 GM 1 binding sites suggests that any differences in delivery of the wt CT-A1 fragment from the cell surface to the cytosol by these holotoxin variants were not rate limiting for development of the morphological manifestations of intoxication.