Testing for prostate-specific antigen (PSA) in blood has enabled early detection of prostate cancer and reduced metastasis and death from disease—but also contributed to overdetection of low-risk cancers. Although no PSA concentration confers zero risk of finding cancer at prostate biopsy, a single PSA measurement at midlife is a remarkably strong predictor of the risk of developing lethal prostate cancer decades later. PSA is a proteolytic enzyme that is non-catalytic in blood, and it occurs in multiple forms. A statistical model based on four kallikrein (4K) markers (free, total, and intact PSA, plus human kallikrein-related peptidase-2 [hK2]) improves specificity in detecting high-grade prostate cancer among men with elevated PSA (reducing unnecessary biopsies) and is also a strong predictor of the risk of lethal prostate cancer decades later. While intra-individual fluctuations in PSA levels are common, an excessive degree of variability is highly problematic, as temporary “false positive” elevations reduce the specificity of PSA as a cancer marker, attenuate the diagnostic value of PSA kinetics, and lead to the use of unnecessary antibiotics. Less studied but similarly abundant in prostatic fluid as PSA, the concentration of microseminoprotein-ß (MSP, MSMB) in blood is inversely associated with prostate cancer risk, and a single nucleotide polymorphism (SNP, rs10993994) in the promoter region of the MSMB gene is also associated with prostate cancer risk, but the role of these markers in clinical decision-making is unclear. Similarly, a SNP in the SERPINA3 gene is significantly associated with blood levels of PSA, and the encoded protein, alpha-1-antichymotrypsin (ACT), is the predominant stable complexing ligand to PSA in the blood. However, the clinical value of these makers is undetermined, and it remains unclear whether ACT levels in blood influence the predictive value of a baseline PSA value or affect intra-individual variation in PSA. Additionally, the intra-individual variation of the 4K-panel is currently unknown but could be determined using high-quality serial samples. As the role of these different molecular markers in combined risk-prediction models of aggressive prostate cancer is not well understood, we plan to delineate the influence of intra-individual variability in serial screening samples on clinical decision- making for risk stratification and biopsy by a single PSA value and additional markers. Using blood samples from the PLCO, Göteborg-1 & -2 trials, and Multiethnic Cohort (MEC), we plan to: 1) quantify the patterns of variation in the 4K markers + MSP in serial measurements; 2) determine the relationship between a statistical model based on 4K markers + MSP and subsequent risk of lethal prostate cancer, then independently validate the clinical utility of the markers in decision-making and risk stratification before treatment decisions in a randomized trial of prostate cancer treatments (ProtecT); and 3) compare head-t...