EnVision+ (EV+, Dako, Copenhagen, Denmark) is a novel two-layer dextran polymer visualization system for immunohistochemistry. We compared EV+ with two sensitive three-layer techniques: an avidin-biotin complex (ABC) system (Vectastain Elite ABC Kit [VABC], Vector, Burlingame, CA, U.S.A.) and a labeled streptavidin-biotin (LSAB) system (nonkit; Dako). Sections from multitissue and other paraffin blocks of formalin-fixed tissues were stained and 10 selected antibodies applied. Furthermore, in a comparison of EV+ and LSAB, 100 of the most frequently used antibodies were applied. In most of the cases, EV+ and VABC equally enhanced the stainings twofold to fourfold compared with LSAB. After adjusting the primary antibody concentrations, the staining results were practically identical. However, when applying some high-concentration antibodies, a comparable staining intensity with the LSAB system produced unacceptable background staining. With the EV+ technique, the stainings were devoid of false-positive reaction due to endogenous biotin. In a few cases, EV+ produced faint staining of collagen and smooth muscle, as well as faint staining of nuclei in a minority of the cells; however, this never interfered with the interpretation of the stainings. The EV+ system is more expensive than the two other systems; however, because it completely overcomes problems with endogenous biotin, EV+ may allow a more efficient heat-induced epitope retrieval (HIER) and, thus, a further dilution of antibodies. Moreover, savings may be gained from a shorter assay time (40 minutes) and a simplified technique. EV+ appears to be a good alternative to the avidin-biotin-based visualization systems.