Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in many eukaryotic cell lines. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. We found that the regulatory subunits of cyclic AMP-dependent protein kinase are retinoylated in the human myeloid leukemia cell line HL60 and cytokeratins are retinoylated in normal human keratinocytes. We have now shown that the intermediate filament protein vimentin is retinoylated in HL60 cells. We found that a retinoylated protein of Mr 55,000 coeluted on anion exchange chromatography and comigrated on either one- or two- dimensional polyacrylamide gel electrophoresis with a protein that also was stained on immunoblots by an anti-vimentin antibody. Both the Mr 55,000 retinoylated protein and immunoreactive vimentin were associated with cell nuclei isolated by two procedures. A large fraction of this nuclear vimentin was detached during exposure to a nonionic detergent buffer suggesting it was bound to the nuclear envelope. The vimentin remaining with the nuclei was associated with the nuclear matrix and not with the chromatin. Desmin, another intermediate filament protein, was not detached from isolated nuclei during exposure to a nonionic detergent buffer. Desmin was found in both the chromatin and the nuclear matrix fractions, and was not retinoylated. These results raise the possibility that retinoylated vimentin may mediate some of the biological effects of RA, including differentiation.