Prostate-specific antigen (PSA), the tumor marker currently used for prostate cancer (PCa), is not specific enough to distinguish between PCa and benign prostate hyperplasia (BPH). Glycan processing is normally perturbed in tumors, therefore we investigated whether changes in glycosylation of PSA could be useful diagnostic indicators. Previously we determined that the glycosylation of PSA secreted by the tumor prostate cell line LNCaP differs significantly from that of PSA from seminal plasma (normal control). We therefore undertook a detailed glycan analysis of PSA derived from sera from PCa patients and, importantly, established that the glycosylation of the PCa serum PSA was significantly different from the PSA from the LNCaP cell line. In comparison with seminal plasma PSA, the fucose content of PSA from the PCa patient serum was significantly lower and there was a decrease in alpha2,3-linked sialic acid. Differences in the glycosylation of PSA derived from PCa patients' sera, seminal plasma, and LNCaP cells were further established by lectin detection, glycosylation immunosorbent assay, and two-dimensional electrophoresis. We also investigated whether the impact of glycosylation changes initiated by the tumor was reflected in the serum glycome. By comparing the glycans released from the total glycoproteins in PCa patient serum with those of normal serum we found an increase in the proportion of sialyl-Lewis x structures. Further analysis of the glycosylation of PSA from PCa and BPH sera will be required in order to determine the utility of these glycan differences to discriminate specifically between benign and malignant prostate states.