Ampicillin-resistant Hemophilus influenzae had not been identified in Fresno, California, before June 1976. In the 12 months that followed, eight resistant type B strains and three resistant nontypable isolates were cultured from patients treated at two hospitals that provide nearly all of the acute pediatric inpatient care for the area. Two of the resistant strains were obtained from patients with invasive infections and represented 4.2 percent of Hemophilus influenzae isolated from blood, cerebrospinal fluid or joint aspirates during the 12 months. The remaining six resistant type B strains were obtained from 117 patients, and were the predominant organism in cultures of other sites, primarily respiratory secretions. In two of three patients infected with nontypable organisms, resistance appeared to emerge during therapy with ampicillin. Measurement of beta lactamase was a practical and accurate method for differentiating between ampicillin-sensitive and resistant strains. All ten of the beta lactamase-positive isolates tested had minimal inhibitory concentrations (MIC) for ampicillin of 15 mug per ml, or less. In contrast 30 beta lactamase-negative strains had MIC's of 1.5 mug per ml, or less, of ampicillin. Our results indicate that ampicillin resistance has become a significant problem in the Central Valley of California and probably the entire state.