The prevalence and clinical implications of colonization with nontuberculous mycobacteria were prospectively studied in 37 patients who had cystic fibrosis. Sputum samples were cultured on Coletsos and Löwenstein-Jensen selective media after decontamination with sodium hydroxide and oxalic acid. Oxalic acid-decontaminated fractions were also cultured in selective liquid medium. Nontuberculous mycobacteria were isolated from 6 patients (16.1%). Mycobacterium chelonae and Mycobacterium avium-intracellulare complex were the most common species. Three patients with positive results of culture had at least 1 positive result by acid-fast smear. Oxalic acid decontamination and culture in liquid medium had the lowest contamination rate (6.7%). Colonization with nontuberculous mycobacteria was associated with humoral response to mycobacteria (immunoglobulin G titers against antigen A60) in patients with samples that tested positive by acid-fast smear. An improvement in pulmonary function was observed in 2 patients after they received a course of antimycobacterial therapy. Screening for nontuberculous mycobacteria in patients with cystic fibrosis will contribute to understanding the relevance of these pathogens with regard to deterioration of pulmonary function in patients with cystic fibrosis.