In Drosophila, a single copy of the gene encoding the phosphoprotein Dishevelled (Dsh) is found. In the genomes of higher organism (including mammals), three genes encoding isoforms of Dishevelled (Dvl1, Dvl2, and Dvl3) are present. In the fly, Dsh functions in the Wnt-sensitive stabilization of intracellular β-catenin and activation of the Lef/Tcf-sensitive transcriptional response known as the Wnt “canonical” pathway. In this study we explore the expression of Dvls in mammalian cells and provide an estimate of the relative cellular abundance of each Dvl. In mouse F9 cells, all three Dvls are expressed. Dvl2 constitutes more than 95% of the cellular pool, the sum of Dvl1 and Dvl3 constituting the remainder. Similarly, Dvl2 constitutes more than 80% of the Dvl1-3 pool in mouse P19 and human HEK 293 cells. Suppression of cellular protein expression can be catalyzed by use of small interfering RNA (siRNA) that target specific protein mRNA. siRNA-induced knock-down of individual Dvls was performed and the Wnt3a-sensitive canonical pathway in F9 cells employed as the read-out. Activation of the canonical signaling pathway by Wnt3a was dependent upon the presence of Dvl1, Dvl2, and Dvl3, but each to a variable extent. Wnt3a-sensitive canonical transcription was suppressible, by knock-down of Dvl1, Dvl2, or Dvl3. Conversely, the overexpression of any one of three Dvls individually was found to be capable of promoting Lef/Tcf-sensitive transcriptional activation, in the absence of Wnt3a, i.e., overexpression of Dvl1, Dvl2, or Dvl3 is Wnt3a-mimetic. Graded suppression of individual Dvl isoforms by siRNA was employed to test if three Dvls could be distinguished from one another with regard to mediation of the canonical pathway. Canonical signaling was most sensitive to changes in the abundance of either Dvl3 or Dvl1. Changes in expression of Dvl2, the most abundant one of the three isoforms, resulted in the least effect on canonical signaling. Dvl-based complexes were isolated by pull-downs from whole-cell extracts with isoform-specific antibodies and were found to include all three Dvl isoforms. Rescue experiments were conducted in which depletion of any one of three Dvl isoforms suppresses Wnt3a activation of the canonical pathway and the ability of a Dvl isoform to rescue the response was evaluated. Rescue of Wnt3a-stimulated transcriptional activation in these siRNA-treated cells occurred only by the expression of the very same Dvl isoform depleted by the siRNA. Thus, Dvls appear to function cooperatively as well as uniquely with respect to mediation of Wnt3a-stimulated canonical signaling. Dvl3 plays the most obvious role, whereas the most abundant Dvl (i.e., Dvl2) plays the least obvious role. These observations suggest that individual Dvl isoforms in mammals operate as a network with some features in common and others rather unique.