AbstractIn this short note, the key points of the histogram analysis method proposed by Watson (Cytometry 2001;43:55–68) were examined. The complete relationship among the mean values of the distributions on which that method is based was derived analytically. It was shown that the symmetry assumption is not necessary, and that the assumption of equal variances of the unlabeled and labeled cell distributions cannot be verified a posteriori. Moreover, a possible shift between the unlabeled cell distribution in the test and the negative control may greatly influence the estimation of the labeled cell percentage. Finally, by using an experimental set of histograms, it was shown that the assumptions underlying Watson's analytical method are likely not to be fulfilled in real immunofluorescence data. © 2009 International Society for Advancement of Cytometry