The biophysical and biochemical properties of Penicillium cyaneo-fulvum virus (Pc-fV) and Penicillium chrysogenum virus (PcV) have been compared. In sucrose density gradient sedimentation, purified virus preparations gave one major component, L, and three minor components E1, E2 and H with sedimentation coefficients of 145S, 80S, 102S and 172S respectively in each case. E1, E2 were shown to be empty particles. Pc-fV L particles contained only double-stranded RNA, which separated in polyacrylamide gel electrophoresis into four components with mol. wt. 2-21 X 10(6), 2-08 X 10(6), 1-98 X 10(6) and 1-93 X 10(6). PcV L particles gave three double-stranded RNA components, which were indistinguishable in polyacrylamide gel electrophoresis from the larger three RNA components of Pc-fV. In both viruses each RNA component was separately encapsidated. In both viruses H particles gave rise to the same double-stranded RNA components as their L particles, but contained, in addition, small amounts of single-stranded RNA. Pc-fV and PcV have been shown to consist of isometric particles of the same size and to be serologically related, and the amino acid compositions of their capsid polypeptides were found to be very similar. The capsid polypeptides of the two viruses were examined by SDS-polyacrylamide gel electrophoresis. In each case E2, L and H particles gave one major polypeptide gamma1, with mol. wt. 130000, whereas E1 particles contained one major polypeptide gamma2 with mol. wt. 115000. The mol. wt. of L particles, determined from sedimentation and diffusion coefficients, was 9-8 X 10(6) for both Pc-fV and PcV. The capsid of the L particles of each virus was estimated to contain 60 molecules of polypeptide gamma1.