Because creatine kinase (CK) appears in cardiac lymph after myocardial infarction, this study was undertaken to determine whether lymph inactivates CK in vitro and whether interruption of cardiac lymph flow influences estimation of infarct size based on plasma CK changes in conscious dogs. After the effects of incubation of canine myocardial CK in native, deproteinized, or sulfhydrylfortified lymph and dialysates had been characterized; effects of interruption of cardiac lymph flow on plasma CK time-activity curves after coronary occlusion were assessed in 13 conscious dogs, seven of which had exteriorized occlusive snares around cardiac lymphatics as well as the left anterior descending coronary artery. Native and deproteinized lymph as well as lymph dialysate markedly inactivated CK in vitro with associated nonenzymatically mediated proteolysis detectable on SDS gels. CK released into blood after coronary occlusion compared to myocardial CK depletion was 50% less in dogs with lymphatic occlusion (P< 0.01) although CK loss in the centers of infarcts (73% and 69%) and overall CK depletion (18% and 20%) were similar in the two groups. Based on comparison of observed to projected plasma CK values prior to lymphatic occlusion, significantly less CK appeared in blood when coronary occlusion was followed by lymphatic occlusion (P < 0.01), although the rate of disappearance of CK from the systemic circulation was not altered. Thus, lymph inactivates CK in vitro, and plasma CK time-activity curves after coronary occlusion are influenced considerably by interruption of cardiac lymph flow, a factor that should be incorporated to refine enzymatic estimates of infarct size.