5'-Ethynyl-2'-deoxycytidine (EdC) is a nucleoside analog that inhibits replication of the herpes simplex virus-1 (HSV-1) KOS strain (ID = 0.2 μg/mL). It also reduces virus-induced cytopathogenicity of HSV-1, HSV-2, and vaccinia virus strains in PRK cells (MICs = 0.2-0.4, 1-2, and 5 μg/ml, respectively). EdC is an inhibitor of thymidylate synthetase, selectively reducing DNA incorporation of [1',2'-H]deoxyuridine over [CH-H]deoxythymidine in PRK cells (IDs = 3 and 120 μg/ml, respectively). It inhibits thymidine synthetase in and reduces proliferation of L1210 cells, an effect which is reversed by addition of deoxythymidine (IDs = 4.4 and 1,000 μg/ml, respectively). EdC has been used to monitor DNA synthesis and cellular replication via click chemistry conjugation of the ethynyl group to an azido group of various fluorochromes.