An electrophoretic separation of a previously undetected myosin heavy-chain (HC) isoform in rodent skeletal muscles, designated as HCIID, could be achieved for the first time by the use of polyacrylamide (PAA) gradient gel of Bar & Pette ((1988) FEBS Lett. 235,153-155). However, when used as a conventional method in our laboratory, their gel system seems not to result in reproducible electrophoretograms. This study was undertaken to examine electrophoretic conditions necessary to identify myosin heavy-chain (HC) isoforms, HCI, HCIIA, HCIIB and HCIID, with special reference to HCIIA and HCIID. In an attempt to explore the effect of the pH in the separating gel on electrophoretogram the gels adjusted to pH8.6 were shown to provide the best and most reproducible results. In the case that PAA gradient gels were adopted on the basis of the original method of Bar & Pette, HCIIA and HCIID isoforms were best distinguished by the gels with 4-11%.0 As characterized by its effect on the mobility of HC isoforms, concentrations of glycerol as well as of PPA were found to be a critical factor. In a series of experiments where PAA gradient gels were used, any gels other than those with 25-30 0lycerol gradient did not have a capacity to separate definitely fast type HC isoforms. Other combinations between concentrations of PAA and glycerol were also examined including homogeneous concentrations. In contrast to the report by Bar & Pette, PAA gradient gels were not essential in distinguishing HCIIA from HCIID. Enhanced electrophoretic mobilities existing between HC isoforms were displayed on homogeneous PAA gels rather than on gradient gels.