The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [35S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of 35S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [35S]sulfate incorporation was compared with [14C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with 35S. Redistribution of 14C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [35S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.