The substitution of codon 43 in the gene rpsL is the single most common mutation found in streptomycin-resistant Mycobacterium tuberculosis. The characterization of this mutation has been hampered by the need for prior cultivation of the mycobacteria, the need for DNA sequencing, or both. In this report we describe a simple and culture-independent technique to detect this mutation directly from sputum samples, requiring little more than a polymerase chain reaction (PCR) machine and a simple agarose minigel. There is no need for labeled probes or DNA sequencing. In a preliminary test of feasibility, interpretable results were obtained from all of 16 smear-positive and 1 of 4 smear-negative, culture-positive samples. Two of two samples containing M. tuberculosis with rpsL codon 43 mutations were correctly identified.