Acid-fast bacilli were isolated from lymph nodes of an immunocompetent child presenting with unilateral cervical lymphadenitis. The slowly growing mycobacterium could not be identified by traditional methods. Direct sequencing of the enzymatically amplified 16S rRNA gene revealed a unique sequence belonging to a previously unrecognized mycobacterium. Direct 16S rDNA sequencing enables definitive identification of mycobacterial isolates. The method is useful for rapid recognition of previously unrecognized pathogens.