Primers complementary to conserved sequences of cucumber mosaic virus (CMV) RNA 3 were designed and reverse transcription-polymerase chain reaction was directly performed in crude sap extracts of CMV-infected plants. All isolates assayed belonging to subgroups I or II from different Spanish geographical locations and infecting tobacco, tomato and cucumber plants were detected. No amplification was observed in healthy or tomato aspermy virus (TAV)-infected plants, nor in CMV-infected pepper plants. A dilution as low as 10(-7) in tobacco crude sap extracts or 100 fg of purified virus (c. 50 000 virus particles) could be detected, results showing that this procedure is 10? times more sensitive than enzyme-linked immunosorbent assay (ELISA).