The mechanism of action of a new anti-rhinovirus compound, dichloroflavan, was examined using a variety of techniques. Dichloroflavan was shown to bind to the highly sensitive rhinovirus type 1B (50% inhibitory concentration 0.007 microM) and at a much lower level to the insensitive rhinovirus type 4 (50% inhibitory concentration greater than 25 microM). Binding of the compound to rhinovirus 1B was accompanied by a reduction (about 0.5 log10 units) in virus infectivity which could be restored by treatment with chloroform. Maximum inhibition of virus yield (about 2 log10 units) occurred only when compound and virus were added together, but some inhibition (about 0.7 log10 units) occurred even when the compound was added near the end of a single cycle of replication. This late reduction in infectivity could be abolished by treating with chloroform, and was therefore caused by the compound binding to progeny virus. The compound did not interfere with adsorption of virus to cells, nor with uncoating of the viral RNA. However, little or no viral RNA and protein synthesis occurred in the presence of dichloroflavan provided that the compound was added with the virus. Addition of the compound after virus adsorption had no effect on either viral RNA or protein synthesis. These results indicate that dichloroflavan binds directly to rhinovirus 1B and appears to prevent virus replication at a point immediately after uncoating of the viral RNA.