ABSTRACT: Beta-microseminoprotein (MSP) is one of the most abundant proteins secreted by the prostate gland. Because MSP is also synthesized in nonreproductive organs, the establishment of a solid relationship between the levels of MSP in serum and semen is crucial for future studies connecting MSP with aging or diseases of the prostate gland. We developed a specific, competitive, europium-based immunoassay to measure MSP in serum and seminal plasma. We also produced recombinant MSP in insect cells using baculo virus and purified it to homogeneity by a novel approach with ethanol extraction and gel filtration. The median values of MSP in 205 young men were 12 μg/L (2.5–97.5 percentile, 4.9–26 μg/L) in serum and 0.53 g/L (2.5–97.5 percentile, 0.13–2.0 g/L) or 1.8 mg (2.5–97.5 percentile, 0.32–6.6 mg) in seminal plasma. MSP in serum showed significant correlation to MSP in seminal plasma (r = .50, P < .001). Significant correlations were also found in seminal plasma between MSP and prostate-specific antigen (PSA) (r = .65, P < .001) and between MSP and Zn2+ (r = .54, P < .001). The yield of recombinant MSP in culture medium was 35 mg/L or higher, and recovery following ethanol extraction was 80%–90%. MSP in serum reflects the prostate secretion of MSP, and correlations were also found in seminal plasma between MSP and PSA and Zn2+. This suggests that MSP in serum can be used as a marker of prostate secretion, despite the contribution from extra prostatic tissues.