We have investigated the potential antiviral activity of three cobalt(III) compounds. Two compounds, Co(III)-cyclen-methylbenzoic acid and its methyl ester derivative, are based on the macrocyclic chelator, cyclen, and were synthesized in our laboratory. Both compounds have been shown to bind tightly to nucleic acids and to hydrolyze phosphodiester bonds. However, neither compound exhibited any significant antiviral activity in an in vitro model of Sindbis virus replication. In contrast, a third compound, Co(III)hexammine, significantly inhibited Sindbis virus replication in baby hamster kidney (BHK) cells in a dose- and time-dependent manner. In plaque assays, the incubation of Co(III)hexammine with Sindbis virus resulted in a dose-dependent decrease in virus replication when measured at both 24 and 48-h post-infection. Over the concentration range of 0-5mM Co(III)hexammine, the IC(50) for the inhibition of viral replication was determined to be 0.10+/-0.04mM at 48h. Additionally, when BHK cell monolayers were pretreated with Co(III)hexammine for 6h prior to Sindbis infection, optimal cellular morphology and plasma membrane integrity were observed at 0.6-1.2mM Co(III)hexammine. Analysis by flow cytometry confirmed that Co(III)hexammine mediated a concomitant dose-dependent increase in BHK cell viability and a decrease in the percentage of Sindbis virus-infected cells (IC(50)=0.13+/-0.04mM). Our findings demonstrate for the first time that Co(III)hexammine possesses potent antiviral activity. We discuss our findings within the context of the ability to further functionalize Co(III)hexammine to render it a highly specific antiviral therapeutic reagent.