A total of 169 strains of Yersinia spp. were analyzed for their ability to grow on two different kinds of cefsulodin-Irgasan-novobiocin (CIN) agar containing 15 or 4 micrograms of cefsulodin per ml, on salmonella-shigella agar, and on MacConkey agar. CIN media inhibited the growth of Yersinia pseudotuberculosis and Yersinia enterocolitica biotype 3B serotype O3 (3B/O3) but not the growth of the other Yersinia organisms used. Relative to growth on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 6% yeast extract, 48 and 44% of Y. pseudotuberculosis and Y. enterocolitica 3B/O3 strains, respectively, were inhibited on CIN I agar (low cefsulodin concentration), and 83 and 54%, respectively, were inhibited on CIN II agar (high cefsulodin concentration) after incubation for 24 h at 32 degrees C. The inhibition of Y. pseudotuberculosis growth was significantly more extensive on CIN II agar than on CIN I agar. The MICs of cefsulodin and novobiocin clearly indicated a higher susceptibility for Y. pseudotuberculosis than for the other Yersinia organisms at 32 degrees C. All Y. pseudotuberculosis strains were susceptible to cefsulodin at 15 micrograms/ml (the approximate concentration used in CIN II agar). Y. enterocolitica 3B/O3 strains were resistant to cefsulodin, Irgasan, and novobiocin at the concentrations used in CIN media. These findings show that cefsulodin inhibits the growth of Y. pseudotuberculosis at the concentration used in CIN media and that growth inhibition of Y. enterocolitica 3B/O3 is related to a component of the CIN Base.