Pig liver GDP-mannose pyrophosphorylase was purified 5,000-fold to apparent homogeneity using standard techniques. The native enzyme showed a single band on gels of about 450 kDa and two subunits of 43 and 37 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 37-kDa (beta-) subunit had only methionine at its amino terminus and a surprisingly hydrophobic sequence: Met-Lys-Ala-Leu-Ile-Leu-Val-Gly-Gly-Tyr-Gly-Thr-Arg-Leu- Arg-Pro-Leu-Thr-Leu-Ser-Ile-Pro-Lys. The 43-kDa (alpha-) subunit was blocked at the amino terminus, but a 29-kDa CNBr fragment had the following sequence: Leu-Asp-Ala-His-Arg-His-Arg-Pro-His-Pro- Phe-Leu-Leu-. Substrate specificity studies done in the direction of formation of nucleoside triphosphate and sugar-1-P indicated that the enzyme was most effective with GDP-glucose as substrate (100%) followed by IDP-mannose (72%) and then GDP-mannose (61%). That GDP-mannose and GDP-glucose activities were indeed catalyzed by the same enzyme was indicated by the following. (i) Various studies indicated that the enzyme was homogeneous. (ii) A staining procedure for production of GTP stained the same single band on native gels when either GDP-mannose or GDP-glucose was the substrate. (iii). GDP-mannose inhibited the utilization of GDP-glucose by the enzyme, and vice versa. When 8-azido-[32P]GTP was incubated with native enzyme and exposed to UV light, both the 43-kDa and the 37-kDa subunits became labeled, although the 37-kDa subunit reacted more strongly. On the other hand, 8-azido-GDP-[32P]mannose only photolabeled the 43-kDa band. Most importantly, the purified enzyme can be utilized to produce 8-azido-[32P]GDP mannose or 8-azido-[32P]GDP glucose.