The protozoan Giardia lamblia is a major cause of parasite-induced diarrhea in humans. Humoral immunity has been shown to be important for clearance of the infection, but only a few antigens have been identified. In this study, we focused on the immunoreactivity of nonvariant antigens. Serum samples from 93 patients with acute giardiasis who were infected during a waterborne outbreak in a nonendemic country were screened on 1-dimensional Western blots. Representative serum samples that reacted strongly with proteins of different molecular weights were further analyzed on 2-dimensional Western blots. Sixteen immunoreactive proteins were identified using mass spectrometry analysis, among them variable surface proteins, alpha-giardins, arginine deiminase, ornithine carbamoyl transferase, and fructose-1,6-bisphosphate aldolase. Several of the identified proteins were immunoreactive in recombinant form, and they may be important in the development of new diagnostic tools and vaccines.