This study investigated the release of antibiotics in vivo, from an articulating polymethylmethacrylate (PMMA) spacer used in two-stage revision arthroplasty of infected hip implants. Forty-six patients who underwent two-stage revision hip arthroplasty for infections were managed with an interim PMMA spacer loaded with a high dose of vancomycin and aztreonam. Serum and aliquots of drainage collected after the first-stage surgery, and joint fluid obtained at the time of the second-stage surgery were analyzed for antibiotic concentrations by high performance liquid chromatography and bioactivity by tube dilution bioassay. Following implantation, the highest levels of antibiotics were measured in aliquots of drainage on the first day (vancomycin: 1538.0±243.6 μg/mL; aztreonam: 1003.5±323.5 μg/mL), decreasing to 571.9±169.4 μg/mL for vancomycin and 313.6±88.3 μg/mL for aztreonam after 7 days. Antibiotic concentrations in serum were very low (vancomycin: 0.58±0.2 μg/mL, range: 0.1-1.6 μg/mL; aztreonam: 0.46±0.3 μg/mL, range: 0.1-0.9 μg/mL at 24 h) and there was no systemic adverse effect. At a mean 107 days after the first-stage surgery, the concentrations of antibiotics in joint fluid were well above the minimal inhibitory concentration of most common microorganisms. The released antibiotics were bioactive against the test organisms. Based on the observed results, we confirmed the safety and effectiveness of in vivo drug delivery from antibiotic-impregnated PMMA hip spacers.