Abstract: This study was undertaken to determine the in vitro activity of ampicillin against β-lactamase producing enterobacterial species. The study was conducted during the period December 2006-August 2009. Two hundred patients were investigated. Clinical specimens collected were 38.5% ear swabs, 36.5% wound swabs and 25% urine specimens. Cultivation of these specimens on enriched and/or differential media yielded 189 bacterial isolates, of which 96 were enterobacterial species. Among the enterobacterial isolates 62.5% were β-lactamase producers as indicated by β-lactamase acidometric method. Ampicillin activity against β-lactamase producers was determined by modified Kirby-Bauer Disc Diffusion technique. The technique was judged and approved by the National Committee for Clinical Laboratory Standards (NCCLS). The results showed that positive β-Lactamase isolates were E. coli 21.6%, K. pneumoniae 15.5%, P. mirabilis 13.7% and P. vulgaris 0.9%. The result indicated that only 8.6% of the β-lactamase producers were sensitive to ampicillin while 91.4% were resistant. It has been concluded that further studies are needed to validate the usefulness of ampicillin in treatment of enterobacterial infections in Sudanese patients.