Anti-carbohydrate monoclonal antibodies (mAbs) are very useful in the functional analysis of complex carbohydrates in vivo. However, such mAbs are difficult to generate, largely because a wide variety of complex carbohydrates is intrinsically expressed in mice and rats and because the antigenicities of glycans are generally poor. In this chapter, I describe an efficient method for generating anti-carbohydrate mAbs using glycan--synthesizing enzyme-knockout mice in which the glycan structures formed by the missing enzymes should be highly antigenic. As an application of this method, I describe the generation of anti-sulfated glycan mAbs using sulfotransferase-deficient mice and the immunohistochemical detection of sulfated glycans involved in lymphocyte homing in both humans and mice.