Five spin-labeled 9-aminoacridines, each bearing either a 4-(2,2,6,6-tetramethyl-1-piperidinyloxy) or a 3-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety in the 9 position, have been synthesized and assayed for biological activity in three different test systems. Sedimentation velocity measurements indicated that the labels caused unwinding of calf thymus DNA. Those acridines which contained both 6-chloro and 2-methoxy substituents were less toxic to leukemia L1210 in static culture than the corresponding unsubstituted analogues. While the unsubstituted aminoacridines were quite good inhibitors of Escherichia coli DNA-primed RNA polymerase, the 6-chloro-2-methoxy-substituted compounds stimulated this enzyme system. In the presence of E.coliDNA, the ESR spectrum of 4-[(6-chloro-2-methoxy-9-acridinyl)amino]-2,2,6,6-tetramethyl-1-piperidinyloxyl (12) became broad and highly asymmteric with a maximal hyperfine splitting of 57.5 G. This observation suggests that when 12 intercalates into DNA the piperidinyl moiety that bears the nitroxide group becomes highly immobilized. These results suggest that the spin-labeled 9-aminoacridines will be useful probes for nucleic acids.