The effect of para-JEM(R) BLUE on Mycobacterium avium subspecies paratuberculosis (MAP) inoculated into broth-based culture media was evaluated by using 84 fecal samples with known MAP status. Results showed that growth of the organism in samples inoculated into the broth without the para-JEM BLUE was detectable 1-35 days (average of 6 days) earlier in 35 of the samples (42%) compared with the same samples inoculated in broth with para-JEM BLUE. Four additional samples (5%) that were MAP positive in the culture broth that lacked the para-JEM BLUE gave negative results when the reagent was included. Of the remaining 45 samples, growth of MAP was detected 1-4 days (average of 3 days) earlier in 4 of the samples (5%) inoculated in the broth with para-JEM BLUE compared with the same samples inoculated in the broth without the para-JEM BLUE, whereas 41 samples (49%) yielded equivalent results with respect to time-to-growth detection and negative growth, regardless of whether para-JEM BLUE was present in the culture broth. However, exclusion of para-JEM BLUE from the broth increased the number of samples that produced false-positive instrument signals compared with the number that produced false-positive signals when the reagent was added. Modification of the sample processing step had no measurable effect. Observations indicated that, although elimination of para-JEM BLUE from the broth increased false-positive instrument signals, its inclusion has an adverse effect on the growth of certain MAP, which suggests that its elimination from broth cultures may increase sensitivity.