Cucumber mosaic virus Fast New York strain (CMV-Fny) containing a mutated 2b protein (CMV-FnyΔ2b) was evaluated for the ability to infect ‘Calwonder’ bell pepper (Capsicum annuum) plants in comparative tests with the parent virus, CMV-Fny. Plants inoculated with CMV-FnyΔ2b did not develop local or systemic symptoms of infection, whereas CMV-Fny-infected plants developed systemic chlorosis by 7 days post inoculation (dpi), followed by mosaic and leaf deformation. Virus accumulation, determined by enzyme-linked immunosorbent assay (ELISA), revealed that CMV-FnyΔ2b accumulated in inoculated Calwonder leaves and inconsistently infected some noninoculated leaves at a low titer but was not detected in the youngest, noninoculated leaves. Immuno-tissue blot tests did not detect CMV-FnyΔ2b in the stems of infected plants, whereas CMV-Fny accumulated throughout the length of the stems of inoculated plants. In two experiments, protoplasts were isolated from Calwonder leaves, inoculated with viral RNAs of CMV-Fny or CMV-FnyΔ2b, and tested by ELISA for infection. In both experiments, less CMV-FnyΔ2b than CMV-Fny accumulated in protoplasts. These results suggest that the CMV 2b protein is needed for systemic infection of Calwonder pepper plants and for accumulation of the virus in inoculated protoplasts.