Background　Hydrolysis　of　beta-lactam　antibiotic　by　beta-lactamase　is　the　most　common　mechanism　of　beta-lactam　resistance　in　clinical　isolates.　Timely　detection　and　characterization　of　beta-lactamases　are　therefore　of　utmost　biomedical　importance.　Conventional　spectrophotometric　method　is　time-consuming　and　cannot　provide　thermodynamic　information　on　beta-lactamases.　Methods:　A　new　assay　was　developed　for　the　study　of　beta-lactamase　activity　in　protein　solutions　(Metallo-beta-lactamase　L1)　and　in　clinical　bacterial　cells,　based　on　heat-flow　changes　derived　from　enzymatic　hydrolysis　of　beta-lactams　using　isothermal　titration　calorimetry.　Results:　(1)　The　thermokinetic　parameters　of　three　antibiotics　(penicillin　G,　cefazolin　and　imipenem)　and　the　inhibition　constant　of　an　azolylthioacetamide　inhibitor　were　determined　using　the　calorimetric　assay.　The　results　from　the　calorimetric　assays　were　consistent　with　the　data　from　the　spectrophotometric　assay.　(2)　The　values　of　heat　change　in　the　calorimetric　assay　using　two　clinical　Escherichia　coli　strains　correlated　well　with　their　antibiotic　susceptibility　results　from　the　broth　dilution　experiment.　The　subtypes　of　beta-lactamase　were　also　determined　in　the　calorimetric　assay.　Conclusions:　The　ITC　assay　is　a　reliable　and　fast　method　to　study　beta-lactamase　enzyme　kinetics　and　inhibition.　It　can　also　provide　thermodynamic　information　on　antibiotic　hydrolysis,　which　has　been　taken　advantage　of　in　this　work　to　study　beta-lactamase　activity　in　two　clinical　Escherichia　call　isolates.　General　significance:　As　the　first　calorimetric　study　of　beta-lactamase　activity,　it　may　provide　a　new　assay　to　assist　biomedical　validation　of　new　beta-lactamase　inhibitors,　and　also　has　potential　applications　on　rapid　antibiotic　susceptibility　testing　and　screening　beta-lactamase　producing　bacteria.