Development of Gram-negative rods resistance to the third generation cephalosporins (cefotaxime, ceftazidime, cefoperazone) in connection with their application at the University Hospital in Olomouc was evaluated in this study. The highest increase in resistance to cefotaxime was detected in Enterobacter cloacae (from 22.9% in 1995 to 49.0% in 1997) and Enterobacter agglomerans strains (28.0% - 40.5%). In addition, increased resistance to ceftazidime in Acinetobacter baumannii (12.5% - 35.1%), Enterobacter aerogenes (7.4% - 20.9%), Enterobacter cloacae (16.7% - 47.2%) and Pseudomonas aeruginosa (4.0% - 26.3%) was observed. Finally, the greatest increase in frequency of strains resistant to cefoperazone was observed in E. aerogenes (18.4% - 30.1%), E. agglomerans (31.0% - 52.3%), E. cloacae (35.5% - 47.2%) and Providencia rettgeri (26.5% - 53.2%). A 23.5% increase in third generation cephalosporin use was evident by evaluation of RDDD(ATB) parameters in 1996 and 1997. Corresponding values for individual antibiotics were 26.5% cefotaxime, 20.7% ceftriaxone, and 40.3% ceftazidime increase. However, cefoperazone use decreased by 10.9%.