The use of proteolytic enzymes has been established in the non-antibiotic treatment of mastitis in dairy cattle. The objective of this study was to evaluate, if enzymes are efficacious in the treatment of chronic endometritis. In a controlled field trial, cows with vaginal discharge 21-27 days in milk (DIM) were randomly assigned to two treatment groups. Endometritis was classified into three categories, depending on the type of vaginal discharge: clear mucus with flakes of pus (E1), mucopurulent discharge or fluctuating contents in the uterus (E2), and purulent discharge (E3). In group ENZYMES (n=191), cows received an intrauterine treatment with a salve containing the enzymes trypsin (16 mg), chymotrypsin (16 mg), and papain (8 mg). Cows in group PGF (n=225) were treated with 0.5mg of cloprostenol. Cows that did not show any clinical signs of chronic endometritis were regarded as healthy control group (HC, n=699). In groups ENZYMES and PGF, all cows were re-examined 35-41 DIM. In group ENZYMES, cows were re-treated with enzymes if signs of endometritis were found, while in group PGF all cows received a second dose of cloprostenol, regardless of their clinical findings. Cure rate after the first treatment, defined as the absence of vaginal discharge at the re-examinations, was 59.7 and 68.0% in groups ENZYMES and PGF, respectively (P>0.05). Reproductive performance measures showed no significant differences between the two treatment groups. Service rate was significantly lower for ENZYMES and PGF, respectively, compared to HC. Conception rates to all services and percentages of cows pregnant by 250 DIM were significantly lower in group ENZYMES compared to HC, while no further differences were found between PGF and HC. In both treatment groups, cure rate and reproductive performance measures were better for cows categorized E1 or E2, than for cows categorized E3, respectively. Conception rate to all services for cows with endometritis category E1 was higher in group PGF than in group ENZYMES (P<0.05). The results of this field trial suggest that prostaglandin F(2alpha) is still the treatment of choice for chronic endometritis in dairy cattle.