Acute pain, the most prominent complication of sickle cell disease (SCD), results from vaso-occlusion triggered by sickling of deoxygenated red blood cells (RBCs). Concentration of 2,3-diphosphoglycerate (2,3-DPG) in RBCs promotes deoxygenation by preferentially binding to the low-affinity T conformation of HbS. 2,3-DPG is an intermediate substrate in the glycolytic pathway in which pyruvate kinase (gene PKLR, protein PKR) is a rate-limiting enzyme; variants in PKLR may affect PKR activity, 2,3-DPG levels in RBCs, RBC sickling, and acute pain episodes (APEs). We performed a candidate gene association study using 2 cohorts: 242 adult SCD-HbSS patients and 977 children with SCD-HbSS or SCD-HbSb0 thalassemia. Seven of 47 PKLR variants evaluated in the adult cohort were associated with hospitalization: intron 4, rs2071053; intron 2, rs8177970, rs116244351, rs114455416, rs12741350, rs3020781, and rs8177964. All 7 variants showed consistent effect directions in both cohorts and remained significant in weighted Fisher's meta-analyses of the adult and pediatric cohorts using P, .0071 as threshold to correct for multiple testing. Allele-specific expression analyses in an independent cohort of 52 SCD adults showed that the intronic variants are likely to influence APE by affecting expression of PKLR, although the causal variant and mechanism are not defined.