Since thiols can undergo nitrosation and the cell membrane is rich in thiol-containing proteins, we considered the possibility that membrane surface thiols may regulate cellular entry of NO. Recently, protein disulfide isomerase (PDI), a protein that catalyzes thio–disulfide exchange reactions, has been found on the cell-surface membrane. We hypothesized that cell-surface PDI reacts with NO, catalyzes S-nitrosation reactions, and facilitates NO transfer from the extracellular to intracellular compartment. We observed that PDI catalyzes the S-nitrosothiol–dependent oxidation of the heme group of myoglobin (15-fold increase in the rate of oxidation compared with control), and that NO reduces the activity of PDI by 73.1 ± 21.8% (P < 0.005). To assess the role of PDI in the cellular action of NO, we inhibited human erythroleukemia (HEL) cell-surface PDI expression using an antisense phosphorothioate oligodeoxynucleotide directed against PDI mRNA. This oligodeoxynucleotide decreased cell-surface PDI content by 74.1 ± 9.3% and PDI folding activity by 46.6 ± 3.5% compared with untreated or “scrambled” phosphorothioate oligodeoxynucleotide–treated cells (P < 0.0001). This decrease in cell-surface PDI was associated with a significant decrease in cyclic guanosine monophosphate (cGMP) generation after S-nitrosothiol exposure (65.4 ± 26.7% reduction compared with control; P < 0.05), with no effect on cyclic adenosine monophosphate (cAMP) generation after prostaglandin E₁ exposure. These data demonstrate that the cellular entry of NO involves a transnitrosation mechanism catalyzed by cell-surface PDI. These observations suggest a unique mechanism by which extracellular NO gains access to the intracellular environment.