Bartonella (Rochalimaea) henselae causes cat-scratch disease, bacillary angiomatosis, peliosis hepatis, and fever in humans. B. henselae can be difficult to culture axenically, and as many as 5 weeks may be required before colonies are visible. We compared how different methods of blood collection and handling affect isolation of this pathogen. Blood specimens from B. henselae-infected cats were collected in both EDTA and Isolator blood-lysis tubes and were subsequently plated onto rabbit blood-brain heart infusion agar by using three different schedules: plating immediately, plating after 24 h at 25 degrees C, and plating after 26 days at -65 degrees C. Colonies were counted 14 and 35 days after plating. Blood collected in tubes containing EDTA, frozen at -65 degrees C, and then plated on blood agar yielded a median of 60,000 CFU/ml, compared with 25,333 CFU/ml after collection in the Isolator tubes (P < 0.01). Frozen blood yielded the largest number of B. henselae colonies for any of the schedules tested. These results support previous observations that the Isolator system is more sensitive than tubes containing EDTA for isolation of B. henselae and suggest that, for cat blood, collection in tubes containing EDTA and subsequent freezing may further improve the sensitivity of detection of B. henselae.