Isolation of Haemophilus ducreyi is the only method for the definitive diagnosis of chancroid. Culture on supplemented gonococcal base (GCHgs) or on supplemented Muller-Hinton agar (MHHb) has yielded the best isolation rates. Sheffield media is an alternative to standard media. We compared the isolation rate of H. ducreyi on GCHgs and MHHb to that on the Sheffield media with and without 5% horse blood. Vancomycin (3 mg/L) was added to all media. Of the 87 specimens cultured from patients with genital ulcer disease, 57 (66%) were positive on either GCHgs or MHHb or on both. Twenty six (30%) were positive on GCHgs only, 15 (17%) on MHHb only, and 15 (17%) on both. One culture was positive on Sheffield medium with 5% horse blood; none were positive on Sheffield medium without horse blood. Stock strains showed good growth at 48 hr on GCHgs and MHHb, but no growth at 48 hr and only minimally detectable growth at 72 hr on Sheffield media with horse blood. No growth was detected on Sheffield media without horseblood. Thus optimal culture of H. ducreyi in Kenya requires two media, supplemented gonococcal base and Muller-Hinton agar. Sheffield media is not useful for the primary isolation of H. ducreyi.