The recent identification of a high-level ceftriaxone-resistant (MIC = 2–4 µg/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empiric antimicrobial monotherapy. The penA gene from H041 ( penA41) is a mosaic penA allele similar to mosaic penAalleles conferring intermediate-level cephalosporin resistance (Ceph^I) worldwide, but has 13 additional mutations compared to the mosaic penA gene from the previously studied Ceph^I strain, 35/02 ( penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MIC of ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve the surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction-fragment exchange and site-directed mutagenesis, we identified three mutations - A311V, T316P, and T483S - that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. Mapping these onto the crystal structure of PBP 2 shows that A311V and T316P are close to the active-site nucleophile, Ser310, that forms the acyl-enzyme complex, while Thr483 lies on a loop close to the active site and is predicted to interact with the carboxylate of the beta-lactam antibiotic. These three mutations have thus far only been described in penA41, but dissemination of these in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.