Gross cystic disease fluid protein (GCDFP-15), also known as prolactin-inducible protein (PIP), is a specific breast tumor marker. GCDFP-15/PIP is also identified as gp17 and/or seminal actin-binding protein (SABP) from seminal vesicles and as extraparotid glycoprotein (EP-GP) from salivary glands. It is an aspartyl proteinase able to specifically cleave fibronectin (FN), suggesting a possible involvement in mammary tumor progression and fertilization. Other functions were attributed to this protein(s) on the basis of its ability to interact with an array of molecules such as CD4, actin, and fibrinogen. We investigated the structure of the protein(s) under disease versus physiological conditions by RP-HPLC chromatography, ProteinChip technology, and QStar MS/MS mass spectrometry. The proteins behaved differently when examined by RP-HPLC chromatography and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry, suggesting different conformations and/or tissue-specific posttranslational modifications of the proteins, although their primary structure was identical by MS/MS analysis. Both showed a single N-glycosylation site. A different N-linked glycosylation pattern was observed in pathological GCDFP-15/PIP as compared with physiological gp17/SABP protein by coupling enzymatic digestion and ProteinChip technology. Furthermore, taking advantage of ProteinChip technology, we analyzed the interaction of both proteins with CD4 and FN. We observed that the physiological form was mainly involved in the binding to CD4. Moreover, we defined the specific FN binding-domain of this protein. These data suggested that, depending on its conformational state, the protein could differently bind to its various binding molecules and change its function(s) in the microenviroments where it is expressed.