Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding. A similar defective phenotype with respect to particle formation and RNA binding was observed when the basic residues Lys28 and Lys29 in the region connecting both zinc fingers were replaced by alanine. In contrast, mutation of the first cysteine residue in the distal zinc finger had no effect on virion production and allowed substantial RNA binding activity of the mutant NC protein. Moreover, this NC mutant virus exhibited wild-type replication kinetics in the feline MYA-1 T-cell line. Interestingly, amino acid substitutions disrupting the highly conserved PSAP and LLDL motifs present in the C-terminus of the FIV NC abrogated virion formation without affecting the NC RNA binding activity. Our results indicate that the proximal zinc finger of the FIV NC is more important for virion production and genomic RNA binding than the distal motif. In addition, this study suggests that assembly domains in the FIV NC C-terminus may be functionally equivalent to those present in the p6 domain of the Gag polyprotein of primate lentiviruses.