The mechanism of the cellular toxicity of four inosinate dehydrogenase (IMP-DH) inhibitors with different antitumor and antiviral pharmacological profiles was investigated in mouse lymphoma (S-49) cell culture. Drug effects on cell growth, nucleotide pools, and DNA and RNA synthesis were measured in the presence and absence of guanine salvage supplies. Both guanine and guanosine were capable of bypassing the IMP-DH block, while they also demonstrated some growth-inhibitory effects when added alone in high concentrations. All four drugs reduced cellular guanosine triphosphate levels and caused secondary changes of the uridine, cytidine, and adenosine triphosphate pools that were similar among the four drugs. However, several drug effects in addition to IMP-DH inhibition were observed except with mycophenolic acid which may represent a pure IMP-DH inhibitor. Both tiazofurin and selenazofurin interfered with the uptake and/or metabolism of uridine and thymidine tracers; however, this effect appeared not to contribute to their cellular toxicity in vitro. Moreover, selenazofurin and tiazofurin impaired the utilization of exogenous guanine salvage supplies for DNA and RNA synthesis, and guanine was particularly ineffective in reversing the toxic effects of tiazofurin on cell growth. This finding is important in view of the available guanine salvage supplies in vivo. Since tiazofurin, selenazofurin, and their known metabolites failed to inhibit hypoxanthine-guanine-phosphoribosyl transferase, guanosine monophosphate kinase, and guanosine diphosphate kinase in cell extracts or permeabilized cells, these drugs may interfere with salvage transport across cellular membranes. The toxic effects of mycophenolic acid and ribavirin were similarly reversed by salvage supplies of up to 200 microM guanine, which suggests that ribavirin primarily acts as an IMP-DH inhibitor under these conditions. This result could explain the rather low antitumor efficacy of both mycophenolic acid and ribavirin in vivo. However, increasing the guanine salvage supply in the medium above 200 microM further reversed the toxic effects of mycophenolic acid to maximum rescue, while it increased the toxicity of ribavirin (300 microM). This finding suggests the presence of a toxic mechanism of ribavirin at higher concentrations that is dependent upon the presence of guanine supplies sufficient to fully overcome the IMP-DH inhibition. This study documents that each antimetabolite displays a unique spectrum of activities with multiple toxic targets.