The pleuromutilin antibiotic tiamulin (TIA) is known to produce a negative interaction in broilers when administered in combination with several ionophore anticoccidials such as salinomycin (SAL). Chlortetracycline (CTC), when administered simultaneously with TIA, has demonstrated a synergistic antimicrobial effect. A 35-d feeding study was conducted in cages to evaluate the interaction effect of a combination of TIA plus CTC at increasing inclusion levels when administered concurrently with SAL. A total of 200 one-day-old broiler chicks were distributed into 4 groups, and each group consisted of 5 cages containing 10 birds in each. Replicate cages were distributed randomly. Feed for all groups contained 60 ppm SAL, but additionally, 0, 20, 30, and 50 ppm TIA and 0, 60, 90, and 150 ppm CTC were included, respectively. Several enzymes (creatine phosphokinase, lactate dehydrogenase, and aspartate aminotransferase) were determined from blood samples taken at the end of the trial. Blood samples were also collected during d 0, 19, and 35 and were analyzed for antibody titers against Mycoplasma gallisepticum and Mycoplasma synoviae. Necropsy of a few birds (20, 8, 20, 12, and 12 on d 7, 14, 21, 28, and 35, respectively) was conducted at weekly intervals. Results indicated that there was a significant depression of weight gain (P < 0.05) in group 4 (TIA 50 + CTC 150) only. The final weights were 1,809 +/- 130, 1,859 +/- 52, 1,703 +/- 47, and 1,617 +/- 98 g for groups 1 (TIA 0 + CTC 0), 2 (TIA 20 + CTC 60), 3 (TIA 30 + CTC 90), and 4 (TIA 50 + CTC 150), respectively. However, feed intake and feed conversion efficiency (g of weight gain/kg of feed intake) were not significantly affected in any of the groups. There was no dose-related adverse effect on mortality or clinical signs exhibited during the trial, and this was supported by necropsy. Maternally derived antibodies against M. gallisepticum were present at the beginning of the trial but disappeared within 19 d. Otherwise, there was no apparent infection by M. gallisepticum or M. synoviae throughout the trial. The results demonstrate that 50 ppm TIA plus 150 ppm CTC along with 60 ppm SAL caused only a depression of growth, but no adverse signs of interaction were detected. Taking into consideration all the aspects of the cost of production, the 20 ppm TIA plus 60 ppm CTC was the most cost-effective level to administer continuously with 60 ppm SAL via the feed, but it would be important to do an additional study using an artificial infection with M. gallisepticum or M. synoviae to know whether this inclusion rate would be sufficient to protect against an infectious challenge.