Secreted aspartic proteinases (Saps) are important virulence factors during Candida albicans mucosal or disseminated infections. A differential expression of individual SAP genes has been shown previously in a model of oral candidosis based on reconstituted human epithelium (RHE), and in the oral cavity of patients. In this study, the ultrastructural localization of distinct groups of Sap isoenzymes expressed during RHE infection was shown by immunoelectron microscopy using specific polyclonal antibodies directed against the gene products of SAP1-3 and SAP4-6. Large amounts of Sap1-3 antigen were found within C. albicans yeast and hyphal cell walls, often predominantly in close contact with epithelial cells, whereas lower quantities of Sap4-6 were detected in hyphal cells. To elucidate the relevance of the expressed Saps during oral infections, we examined the effect of the aspartic proteinase inhibitor, pepstatin A, during infection of the RHE. The extent of lesions caused by the strain SC5314 was found to be strongly reduced by the inhibitor, indicating that proteinase activity contributes to tissue damage in this model. To clarify which of the SAP genes are important for tissue necrosis, the histology of RHE infection with Deltasap1, Deltasap2, Deltasap3, Deltasap4-6 and three Deltasap1/3 double mutants were examined. Although tissue damage was not blocked completely with these mutants, an attenuated phenotype was observed for each of the single sap null mutants, and was more strongly attenuated in the Deltasap1/3 double null mutants. In contrast, the lesions caused by the Deltasap4-6 triple mutant were at least as severe as those caused by SC5314. During infection with the mutants, we observed that the SAP gene expression pattern of the Deltasap1 and the Deltasap1/3 mutants was altered in comparison with the wild-type strain. Expression of SAP5 was observed only during infection with the Deltasap1/3 mutant, whereas upregulation of SAP2 and SAP8 transcripts was observed in the Deltasap1 and the Deltasap1/3 mutants. These results suggest that Sap1-3, but not Sap4-6, contribute to tissue damage in this model. Furthermore, C. albicans may compensate for the deletion of certain SAP genes by upregulation of alternative SAP genes.