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Objectives: To assess knowledge, diagnostic and treatment practices of the referring private practitioners of Nagpur city regarding Revised National Tuberculosis Control Programme (RNTCP). Methods: The study involved interview of 103 Private Practitioners (PPs) of Nagpur city. Knowledge of private practitioners was assessed based on questions related to diagnosis, categorization, treatment regimens & follow up. Practices of private practitioners were assessed based on which investigations and treatment regimen they advise & whether they offer supervised treatment. Their willingness to get involved in the programme was also recorded. Results: Only 49 (47.6%) private practitioners knew sputum smear examination as primary tool of diagnosis of TB. Only half, 52 (50.5%) of the private practitioners knew number of categories of tuberculosis correctly and 64 (62.1%) private practitioners did know how to categorize TB patients. Chest X-ray and Mantoux test (38.5%) was mainly used by the PPs for TB diagnosis. 42.7% of PPs were prescribing treatment for TB and among them only 8 were prescribing as per RNTCP guidelines and just one provided treatment under direct observation. Different combination of HRZE and HRZES was prescribed for variable period ranges from 2-8 months. And only 12 (11.6%) private practitioners expressed their willingness to get involved in RNTCP for TB control. Conclusion: There is lack of adequate knowledge, diagnostic and treatment practice among PPs as per RNTCP guidelines and further encouragement is required for their participation in the programme.