The cellular contents of protein-bound and nonprotein sulfhydry (-SH) and disulfide (-SS-) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the -SH form. The content of protein-bound groups, and hence the total content of -SH and -SS- groups (28 x 10(-15) moles/cell, or 1.1 x 10(-6) moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 x 10(-10) g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein -SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 x 10(-15) moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total -SH and -SS- content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound -SH groups and mixed -SS- linkages rise to maxima while protein-bound -SS- groups fall to a minimum at the G(1)/S transition, and fluctuations in these groups occur again during G(2). In addition, the protein-bound -SH concentration falls continuously during the S phase. The nonprotein -SH concentration undergoes the largest (relative) fluctuations, dropping from 4 x 10(-15)moles/cell in early G(1) to about 0.4 x 10(-15) moles/cell (of standard protein content) at the end of G(1), and then rising to 30 times this value by the end of S.