Abstract Upon direct selection of mammalian cells for resistance to thapsigargin (TG), a potent inhibitor of the sarcoplasmic/endoplasmic reticulum Ca 2+transport ATPase (SERCA), the ATPase can acquire specific mutations at amino acid position 256 (aa 256). In particular, Phe 256→ Leu and Phe 256→ Ser substitutions can occur upon TG selection, with each substitution resulting in a SERCA that is 4- to 5-fold resistant to TG inhibition (M. Yu et al., J. Biol. Chem.273, 3542–3546, 1998). We have now identified a third substitution, i.e., Phe 256→ Val, that occurs when the Chinese hamster lung fibroblast cell line DC-3F is selected for TG resistance. Although the Phe 256→ Val substitution at codon 256 results in a SERCA whose enzymological properties in terms of Ca 2+transport and ATP hydrolysis are essentially similar to that of wild-type (wt) SERCA, the mutant enzyme is more than 40-fold resistant to TG inhibition. To analyze further the role of aa 256in TG–SERCA interactions, mutational analysis of this particular residue was also carried out. Of all the mutations introduced, only the Phe 256→ Glu substitution interferes with expression of the ATPase. The Phe 256→ Arg substitution does not interfere with SERCA expression, but the resulting enzyme is totally inactive. In terms of sensitivity of the various mutants to TG, maximal reduction in the ATPase's affinity for TG occurs with amino acid substitutions containing branched side chains, i.e. with the Phe 256→ Val, Phe 256→ Ile, and Phe 256→ Thr mutants. Since a corresponding Phe is conserved in the Na +, K +-ATPase which is not sensitive to TG, our findings suggest that this amino acid provides stabilization of the stalk segment with respect to the membrane interface, thereby optimizing specific interactions of TG with neighboring S3 residues (L. Zhong and G. Inesi, J. Biol. Chem.273, 12994–12998, 1998). It is likely that a relatively high frequency of codon 256 mutations favor the aa 256mutants as a specific adaptive response to TG selection.