Gentamicin (G) and vancomycin (V) concentrations in joints fluids obtained from patients during the first 24 hours after implantation of antibiotic-loaded polymethylmethacrylate (PMMA) spacers in two-stage revision for infected arthroplasty, and the inhibitory activity of joint fluids against different multiresistant clinical isolates were studied. A total of 12 patients undergoing two-stage revision surgery with implantation of industrial G spacers added with different amounts of V was studied. Serum and joint fluid samples were collected 1, 4, and 24 hours after spacer implantation. Antibiotics concentrations and joint bactericidal titer (JBT) of combination were determined against multiresistant staphylococcal strains. The local release of G and V from PMMA cement seemed prompt and effective. Serum levels were below the limit of detection. The same joint fluid showed different activity according to the susceptibility of the pathogens tested. Gentamicin and V were released from spacers at bactericidal concentrations exerting a strong inhibition against methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) strains.