D-type cyclins are necessary and rate-limiting for G1 progression during the mammalian cell cycle. Cyclins D1, D2, and D3 are encoded by distinct genes and are expressed in proliferating cells in a lineage-specific manner. Monoclonal antibodies (mAbs) generated to bacterially produced recombinant D-type cyclins were able to react with the native proteins expressed in mammalian cells. One mouse and three rat mAbs immunoprecipitated cyclin D1 from mouse macrophages. Only rat mAbs reacted with human cyclin D1 and cross-reacted with cyclin D2 expressed in proliferating T lymphocytes and human tumor cell lines. A single rat mAb to cyclin D2 exhibited a pattern of reactivity reciprocal to that of rat mAbs to D1. Three rat mAbs reacted specifically with mouse or human cyclin D3, but did not cross-react with cyclins D1 or D2 from either species. Representative mAbs were useful for immunoblotting and detected D-type cyclins coprecipitating in complexes recovered with antiserum to cyclin-dependent kinase-4 (CDK4). Because these mAbs detect D-type cyclins in the nuclei of fixed permeabilized cells, they should prove useful in documenting cyclin overexpression in those human tumors in which the genes are amplified or are targets of specific chromosomal rearrangements.