Lysates of cultured cells have been analyzed for arginine-specific endoproteases using peptidyl-p-nitroanilides as chromogenic substrates. The enzymes present in MDBK, MDCK, VERO, BHK, and chick embryo cells required lysine-arginine or arginine-arginine pairs as cleavage sites, whereas chorioallantoic membrane cells contained, in addition, an activity that could cleave at a single arginine. The effect of peptidyl chloroalkyl ketones on the activation of the fowl plague virus hemagglutinin by the proteases specific for paired basic residues has been investigated. When virions containing uncleaved hemagglutinin were incubated with lysates of uninfected cells, cleavage was completely inhibited by peptidyl chloroalkyl ketones containing paired basic residues at a concentration of 1 mM. In contrast a compound containing a single arginine had no inhibitory activity. When dibasic peptidyl chloroalkyl ketones were added to infected cell cultures, cleavage of hemagglutinin and multiple cycles of virus replication were inhibited at 10 mM. However, a 100- to 200-fold increase of the inhibitory activity in intact cells could be achieved by N-terminal acylation. These studies suggest a potential role of peptidyl chloroalkyl ketones as antiviral agents.