To examine the importance of the nine native cysteine residues in the human A(1) adenosine receptor, each cysteine was individually mutated to both serine and alanine. Saturation binding with the A(1) selective antagonist [(3)H]DPCPX [8-cyclopentyl-1,3-di(2, 3-(3)H-propyl)xanthine] resulted in a wild-type K(d) value of 0.92 nM. All serine and alanine mutants had similar K(d) values with the exception of serine/alanine mutations at Cys80 and Cys169. These two cysteine residues, which are highly conserved in G protein-coupled receptors and hypothesized to be linked through a disulfide bridge, demonstrated no detectable binding with [(3)H]DPCPX. Both serine and alanine mutations at residues Cys80 and Cys169 resulted in receptors that were not detectable at the cell surface, as visualized by immunostaining. The serine/alanine mutants that did bind [(3)H]DPCPX were characterized further through competition binding with the antagonist theophylline and the agonists NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA [(R)N(6)-phenylisopropyl adenosine]. The wild-type theophylline K(i) value was 2.41 microM, with the serine/alanine mutants having similar values. Wild-type NECA and R-PIA K(i) values were 0.74 microM and 97.0 nM, respectively. All mutants had K(i) values similar to wild-type with the exception of the Cys85Ser mutant, which had NECA and R-PIA values of 9.30 microM and 387.3 nM, respectively. These data show that Cys80 and Cys169 are absolutely required for delivery of the receptor to the plasma membrane. The Cys85Ser data indicate that although a cysteine is not required at this position, this residue may have an important role in ligand binding or for the structure of the receptor.