Abstract Tissue plasminogen activator (t-PA) is synthesised as a one chain molecule which can be converted to a two chain form by hydrolysis of the peptide bond after the arginine at position 275 by plasmin. Variants with non-basic residues at position 275 are resistant to this hydrolysis. However, incubation of these variants with plasmin can result in the formation of a ‘two chain’ molecule containing a new amino terminus at position G278. In R275E t-PA, the peptide bond to the carboxyl side of the lysine at position 277 was found to be over 100-fold less sensitive to plasmin than is the peptide bond following the arginine at position 275 in wild-type t-PA. Concomitant with the appearance of the ‘two chain’ variant was a decrease in the ability of the variant to activate plasminogen in the presence of a fibrin(ogen) cofactor. The ability of the ‘two chain’ variant to activate plasminogen in the absence of a cofactor was not decreased as significantly. An R275E/K277I variant of t-PA remained in the single chain form upon plasmin treatment and retained the ability to activate plasminogen in the presence of fibrin to a significantly greater extent than the other position 275 variants.