Cellular ribozymes exhibit catalytic activity in media containing large numbers of anionic compounds and macromolecules. In this study, the RNA cleavage activity of the hammerhead ribozyme induced by Mg2+ was investigated using the solutions containing background nucleic acids, small phosphate and carboxylic acid compounds, and neutral polymers. Analysis of the substrate cleavage kinetics showed that the anionic compounds do not affect the ribozyme activity in Mg2+-saturated solutions and there is almost no effect of the anion–Mg2+ complexes formed. On the other hand, the rate of substrate cleavage in Mg2+-unsaturated solutions was reduced under conditions of a high background of anionic compounds found in cells. The extent of the reduction was more with a greater net negative charge, caused by decreased amounts of Mg2+ that could be used for the ribozyme reaction. It was remarkable that background DNA and RNA molecules having phosphodiester bonds reduced the cleavage rate as much as adenosine monophosphates having a charge of −2 when the effects of the same amount of phosphate groups were compared. Greater reductions in rates than those expected from the molecular charge were also observed in the background of fatty acids that form micelles. An addition of poly(ethylene glycol) to the solutions partially restored the ribozyme activity, suggesting a possible role of macromolecular crowding in counteracting the inhibitory effects of background anions on the ribozyme reaction. The results have biological and practical implications with respect to the effects of molecular environment on the efficiency of ion binding to RNA.