Subforms of creatine kinase, moieties derived from the same isoenzyme but exhibiting slightly different isoelectric points (isoforms), appear in plasma after release of CK isoenzymes from myocardium undergoing infarction. To determine whether isoform patterns in plasma permit precise dating of the onset of initial and recurrent infarction, it is first necessary to characterize the kinetics of isoform interconversion and to ascertain whether one dominant form is present in myocardium prior to release of each tissue isoenzyme. Accordingly, we developed a nondenaturing procedure for quantification of MM CK isoforms and analyzed tissue isoform content and kinetics of isoform interconversion in plasma in vivo and in vitro. MM CK in canine myocardium was found to consist of predominantly one isoform (95%), MMA (pI = 7.91). When purified MMA (420 IU/kg) was injected intravenously in conscious dogs, two isoforms, MMB (pI = 7.74) and MMC (pI = 7.51), appeared with a consistent temporal pattern, and MMA disappeared from plasma within 8 hours, with a disappearance rate three times greater than that of total MM CK activity. Incubation of MMA in vitro at 37 degrees C with canine plasma in concentrations comparable to those after intravenous administration in vivo resulted in a similar temporal pattern of appearance of MMB and MMC and disappearance of MMA with kinetics correlating closely with those in vitro (for MMA disappearance r = 0.985, and for MMC appearance r = 0.986). Incubation of purified MMB and MMC with plasma demonstrated that the conversion of MMA to MMB and to MMC was sequential and unidirectional. Specific activity (international units per milligram immunoassayable protein) was the same for all three isoforms. These results indicate that several conditions necessary for delineation of the chronology of infarction by isoform analysis are fulfilled and that kinetics of interconversion of isoforms in vivo are paralleled in vitro.