Hilsenhoff's biotic index (HBI) is used for evaluating the water quality of Wisconsin streams. The variability of the HBI must be known or estimated in order to detect significant differences in water quality of various streams, at different locations in the same stream, or of the same location sampled at different times. Based on our study results, the best estimate of the HBI standard deviation(s) in fall samples is 0.197. From (s) and variance (s2= 0.0388) estimate we present 2 statistical procedures that test for significant differences between paired HBI values—the detectable difference (DD) method for equal sample sizes (statistically preferred), and the t-test for unequal sample sizes.