To examine how vimentin assembles into the cytoskeletons of cultured cells, we used pulse labeling with [35S]methionine, cell fractionation with Triton X-100, and immunoprecipitation with a monoclonal antibody that binds both nascent and full-length vimentin polypeptides. In embryonic muscle cells, fibroblasts, and erythroid cells, we find two populations of newly synthesized vimentin. One population is found on the cytoskeleton immediately after a 2-min pulse with labeled methionine; the other is delayed in its association with the cytoskeleton and has a measurable rate of disappearance from the extractable pool. This rate varies with cell type, being over 3-fold faster in muscle and fibroblast cells than in erythroid cells. By using [3H]puromycin to specifically label nascent chains, we detect nascent vimentin chains that are bound to the cytoskeleton independently of ribosomes. The fraction of newly synthesized, full-length vimentin that associates with the cytoskeleton immediately correlates in these cell types with the fraction of nascent vimentin chains that are not released from the cytoskeleton by puromycin, RNase, or 0.6 M NaCl. Over one-half of the newly synthesized vimentin associates immediately in muscle and fibroblasts, whereas this value is less than 15% in erythroid cells. These data suggest that the process of vimentin assembly may vary both kinetically and mechanistically in different cell types.