Introduction: Earlier reports indicated that gentamicin should not be mixed with heparin due to precipitation and drug mixture incompatibility. With the recent increased interest in antibiotic lock technique for prophylaxis or adjuvant therapy for hemodialysis catheter-associated infections, it became prudent to reexamine the issue. Method: A mixture of gentamicin (100 µg/mL) and heparin (5,000 U/mL) in normal saline was stored for up to 4 weeks at 4ºC. Gentamicin concentration and bactericidal and anticoagulant activities were measured for up to 4 weeks of storage. Results: The mixture did not show any turbidity or precipitation after fresh preparation, at 48 hours or 4 weeks of storage at 4ºC. Gentamicin concentration (mean ± SD) was 89 ± 7.5 µg/mL, 87 ± 1.0 µg/mL and 85 ± 3.0 µg/mL; gentamicin bactericidal activity was at 1:256 ± 0, 1:256 ± 0 and 1:213 ± 74 dilutions, after fresh preparation, at 48 hours or 4 weeks of storage at 4ºC, respectively (p=NS). Heparin anti-Xa activity was 7,500 ± 990 U/mL and 6,700 ± 707 U/mL, fresh mixture vs. after 4 weeks of storage at 4ºC, respectively (p=NS). Conclusion: In our in vitro experiments we showed that a mixture of gentamicin (100 µg/mL) and heparin (5,000 U/mL) in normal saline retained its gentamicin concentration, bactericidal activity and anticoagulant activity for up to 4 weeks of storage at 4ºC. We also present a brief review of the literature on this subject.