We have reported (R. Balint and S. S. Cohen, 1985, Virology 144, 181-193) that protoplasts from plants infected with turnip yellow mosaic virus (TYMV) continue to produce virus in culture and that newly formed virus particles contained predominantly newly synthesized spermidine and spermine. Inhibition of spermidine synthesis by dicyclohexylamine (DCHA), however, led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virions. We now report similar results with healthy protoplasts infected in vitro, in which essentially all of the virus is newly formed. Again, newly synthesized spermidine and spermine were preferentially incorporated into virus. DCHA inhibited spermidine synthesis by 85%, leading in 20 hr to a 60% depletion of the cellular spermidine and a 30% reduction in the amount of spermidine per virion. Spermine synthesis increased, however, producing a 40% increase in cellular spermine and 50-100% increase in the amount of spermine per virion. Thus, in spite of spermidine depletion, the total positive charge contributed by polyamines to the virus was essentially conserved.