We have used pulse-chase experiments to study the time interval between the synthesis and assembly of tubulin and neurofilament proteins (NFP) in sympathetic neurons grown in tissue culture. After varying pulse-chase times, cultures were extracted with Triton X-100 such that polymerized tubulin and NFP were insoluble, while unassembled tubulin and NFP were quantitatively solubilized. The partitioning of labeled tubulin and NFP between Triton X-100-soluble and insoluble, or cytoskeletal, fractions was determined with an isoelectric focusing X SDS gel electrophoresis assay. Labeled tubulin and NFP in cultures pulse-labeled for 5-10 min partitions primarily with the soluble fraction. When pulse-labeled cultures were chased for increasing periods of time, relatively more of the total labeled tubulin and NFP partitioned with the cytoskeleton, attaining maximal values after chase times of 60-120 and 15-30 min, respectively. The maximal values for the relative levels of labeled tubulin and NFP in polymer were 70-75 and greater than 90%, respectively. The levels of labeled tubulin and NFP synthesized during a short pulse-label remained constant for at least 2 hr, indicating that selective turnover of soluble tubulin and NFP does not detectably contribute to the changes in solubility properties of these proteins observed in the pulse-chase experiments. These results indicate that newly synthesized tubulin and NFP are rapidly assembled from soluble precursors. The lag between the synthesis and assembly of the 145,000-molecular-weight NFP is not related to its phosphorylation because its initial incorporation into the cytoskeleton occurs prior to its phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)