The efficacy of swab transport systems in maintaining viability of Neisseria gonorrhoeae and Streptococcus pneumoniae is crucial both for establishing definitive diagnosis and for monitoring emerging resistance. We tested the efficacy of a newly modified Amies charcoal swab transport system, the StarSwab SP131X (Starplex Scientific, Inc., Etobicoke, Ontario, Canada), by using a combined total of 31 clinical and American Type Culture Collection stock reference strains of N. gonorrhoeae and S. pneumoniae in 46 suspensions of concentrations ranging from 10 5 to 10 8 CFU/ml. Triplicate swabs per strain held at room temperature for 0, 24, and 48 h were plated without prior vortexing, and their growths were graded. All 31 strains were viable at 0 and 24 h. Gonococcal viability at 48 h varied considerably, even among strains with comparable inoculum sizes, suggesting that viability might be strain dependent and confirming the different structural and growth profiles of gonococcal strains. S. pneumoniae strains showed consistent viability, with all strains recovered at all holding periods. This study demonstrates that the StarSwab SP131X is capable of maintaining the viability of N. gonorrhoeae and S. pneumoniae for at least 24 and 48 h, respectively, and reinforces the need for adequate sampling and for timely processing of specimens to maintain optimum performance.