Peptidyl fluoromethyl ketones with the structure carbobenzoxy (Z)-L-phenylalanine-L-alanine-CH2F (MDL 201,053), and its diastereomer Z-L-phenylalanine-D-alanine-CH2F (MDL 201,117), were synthesized and evaluated in vitro for inhibition of purified human cathepsin B. MDL 201,053 was shown to be a potent inhibitor of cathepsin B activity, whereas MDL 201,117 was more than 100-fold less active. In rats with adjuvant induced arthritis, oral administration of MDL 201,053 (13 or 34 mg/kg/day), but not MDL, 201,117 (28 mg/kg/day), significantly decreased the severity of gross clinical arthritis and reduced histologically graded articular cartilage and bone destruction by 76 to 100%. Quantitative image analysis of radiographs indicated that MDL 201,053 treatment significantly reduced bone density changes and inhibited focal bone erosion that normally occur during the course of adjuvant disease. MDL 201,117 had no significant effect on cartilage or bone destruction by any of the evaluation methods used. The effects of MDL 201,053 treatment were dose dependent and treatment was at least partially effective when initiated after the onset of disease. Our studies suggest that inhibitors of cathepsin B may be useful for the treatment of chronic inflammatory joint disease.