Measuring serum levels of the prostate specific antigen (PSA) is the most common screening method for prostate cancer. However, PSA levels are affected by a number of factors apart from neoplasia. Notably, around 40% of the variability of PSA levels in the general population is accounted for by inherited factors, suggesting that it may be possible to improve both sensitivity and specificity by adjusting test results for genetic effects. In order to search for sequence variants that associate with PSA levels, we performed a genome-wide association study and follow-up analysis using PSA information from 15,757 Icelandic and 454 British men not diagnosed with prostate cancer. Overall, we detected a genome-wide significant association between PSA levels and SNPs at six loci: 5p15.33 (rs2736098), 10q11 (rs10993994), 10q26 (rs10788160), 12q24 (rs11067228), 17q12 (rs4430796), and 19q13.33 (rs17632542 (KLK3: I179T), each with Pcombined < 3×10−10. Among 3,834 men who underwent a biopsy of the prostate, the 10q26, 12q24, and 19q13.33 alleles that associate with high PSA levels are associated with higher probability of a negative biopsy (OR between 1.15 and 1.27). Assessment of association between the 6 loci and prostate cancer risk in 5,325 cases and 41,417 controls from Iceland, the Netherlands, Spain, Romania, and the US showed that the SNPs at 10q26 and 12q24 were exclusively associated with PSA levels, whereas the other 4 loci also were associated with prostate cancer risk. We propose that a personalized PSA cutoff value, based on genotype, should be used when deciding to perform a prostate biopsy.