OBJECTIVE To investigate the bacterial flora of the proximal part of the small intestine of healthy cats and determine the effect of sample collection method on results of bacteriologic culture. METHODS 25 healthy barrier-maintained specific-pathogen-free-derived cats. METHODS Aspirated, undiluted samples of proximal small intestinal juice were obtained via oral endoscopy (UEA), and a second sample was collected after instillation of 1 ml of sterile saline solution (diluted, DEA). Undiluted juice also was obtained by direct needle aspiration (NA) from the intestinal lumen. Samples for quantitative and semiqualitative bacteriologic examination were grown aerobically and anaerobically. RESULTS Mean (range) log10 colony-forming units of total bacteria/ml were 6.2 (2.0 to 8.3) for NA, 6.0 (2.0 to 7.9) for UEA, and 4.9 (2.0 to 7.5) for DEA samples. One cat had no growth (< or = 2.0 colony-forming units/ml) for samples obtained using all 3 methods, and another cat had no growth for the DEA sample only. Mean total aerobic, anaerobic, and bacterial counts were not significantly different between NA and UEA methods, but these techniques yielded significantly higher mean counts than did DEA samples (P < or = 0.002, ANOVA). As a percentage of the total bacteria isolated, anaerobes constituted a median 35, 32, and 50% of the NA, UEA, and DEA samples, respectively. Good correlation was found between the NA and UEA samples for total bacteria, aerobes, and anaerobes (r > or = 0.830). CONCLUSIONS Compared with human beings, healthy cats carry high numbers of bacteria in the proximal part of the small intestine. By comparison with NA samples, UEA samples accurately reflected bacterial populations in the small intestine, whereas DEA samples significantly underestimated these populations.