Antimicrobial resistance is an increasing problem in Neisseria gonorrhoeae (NG) treatment. Presently, third-generation parenteral cephalosporins, like ceftriaxone and cefotaxime, are the first option. Resistance to oral, but not to parenteral, third-generation cephalosporins has been reported previously. We analysed the microbial susceptibility (as minimum inhibitory concentration - MIC) of NG cultures obtained from high-risk visitors of the largest Dutch outpatient clinic for sexually transmitted infections (STI) in Amsterdam, the Netherlands. Among 1,596 visitors, we identified 102 patients with at least one NG isolate with reduced susceptibility to cefotaxime (0.125 microg/ml < MIC < or = 0.5 microg/ml). The percentage of NG isolates with reduced susceptibility to cefotaxime rose from 4.8% in 2006 to 12.1% in 2008 (chi2 17.5, p<0.001). With multivariate logistic regression, being a man who has sex with men (MSM) was significantly associated with reduced susceptibility to cefotaxime (p<0.001). Compared to susceptible NG isolates, those with decreased susceptiblity to cefotaxime were more often resistant also to penicillin (16.5% vs. 43.3%), tetracycline (21.5% vs. 68.9%) and ciprofloxacin (44.4% vs. 90.0%, all p<0.001). The increased prevalence of NG strains with reduced susceptibility to cefotaxime among MSM may herald resistance to third-generation parenteral cephalosporins. A considerable proportion of these strains show resistance to multiple antibiotics which could limit future NG treatment options.