The organ distribution pattern of niosome encapsulated rifampicin (5 mg/ml) compared with that of free rifampicin solution was studied in albino rats (n = 30) in order to determine the potential of the delivery system for the site specific delivery of rifampicin, as well as to investigate the most effective route of administration of this delivery system. The comparative area under the curve (AUC) study (t = 0 to 24 h) indicated a higher organ to serum AUC ratio in the case of niosomes (117,060 for lung/serum, 67 for liver/serum and 3,068 for kidney/serum) versus those obtained following the administration of the free drug solution (558.3 for lung/serum, 16.1 for liver/serum and 332.6 for kidney/serum). After intratracheal administration, the lung to serum concentration ratio for niosomes was 128,585 versus 885 for the free drug solution, being a 145 times increase in the drug accumulation capacity of rifampicin niosomes compared with the free drug solution in the lungs. A significant difference in the total drug concentration in the lungs, liver, kidneys and blood serum was found between rifampicin-loaded niosomes and free rifampicin solutions (P < 0.001).