Studies were carried out in human lung fibroblasts (IMR-90) to investigate 1) the relative contribution of two extracellular pools, inorganic sulfate and sulfur-containing amino acids, to the intracellular fraction precipitable by trichloroacetic acid and 2) the possibility that the transport of these sulfur-containing substrates at the plasma membrane may be a limiting step for macromolecular sulfation. Our studies indicate that the ability to use SO42- released by intracellular catabolism of the sulfur-containing amino acid L-cysteine differs from one cell system to another. In contrast to smooth muscle cells, in the human lung fibroblast, L-cysteine contributes significantly to the intercellular pool of SO42- used for sulfation at extracellular [SO42-] < 100 μM. However, under physiological conditions with respect to SO42- ([SO42-]0 = 300 μM), L-cysteine does not contribute >30% of the sulfate incorporated into the cellular fraction. Taurine (2-aminoethanesulfonic acid) inhibits SO42- incorporation into the cell-associated macromolecular fraction. However, results suggest that the effect is not due to either SO42- released by its catabolism or to an effect on SO42- transport into the cell. The fact that the transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits sulfate incorporation indicates that carrier-mediated sulfate transport at the cellular plasma membrane may be a limiting step for sulfate incorporation. In conclusion, under physiological conditions with respect to SO42-, inorganic sulfate is a major source of sulfate for sulfation in human lung fibroblasts and macromolecular sulfation may be limited by its transport into the cells.