Six mutants of alfalfa mosaic virus (AIMV), previously found to have conditionally lethal defects when inoculated to tobacco leaf discs, were assayed for a temperature-sensitive (ts) phenotype in cowpea protoplasts. At 30 degrees the virus production of four mutants was less than 10% of that obtained at 25 degrees , whereas wild-type AIMV multiplied with similar efficiencies at both temperatures. Supplementation experiments performed in protoplasts with two mutants indicated the presence of mutations on both RNA 1 and RNA 2. Viral plus- and minus-strand RNA synthesis induced by these mutants was monitored by the Northern blotting technique under various supplementation conditions. The ts defect was mainly in the production of viral minus-strand RNA whereas transcription of minus-strand into genomic and subgenomic RNAs was much less affected. In addition, results indicate that replication of RNA 1 requires a function not essential for the synthesis of the other RNAs. One of the mutants showed a host-dependent expression of ts mutations, probably reflecting that host-viral interactions play an important role in A1MV replication.