The cause of separation in gel electrophoresis between highly homologous ss (singlestranded) DNAs as observed in SSCP (single-strand conformation polymorphism) was pursued. Advancing our previous explanation [J. Biochem. 99, 663–671 (1986)], the mobility difference of ssDNAs was correlated to differences in their dynamic conformation (not to differences in their most stable structure), focusing on point-substituted sites. The contribution of semistable conformation dynamics was considered to be critical. Putative factors which may influence mobility (i.e., length of ssDNAs, location of substituted sites, and types of substitutions) were experimentally examined and critically discussed. Understanding of these phenomena should yield improvements in various techniques, such as SSCP, and in evaluation of the solution structures of DNA.