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Recent studies have shown that normalization of the lactulose breath test (LBT) with neomycin leads to a significant reduction in irritable bowel syndrome (IBS) symptoms. This subanalysis was done on the constipation-predominant IBS subgroup of patients (C-IBS) to test the ability of neomycin to improve constipation and its correlation with the elimination of methane on breath test. IBS subjects underwent LBT in a blinded fashion. They were then randomly allocated to neomycin or placebo groups. For the purpose of this analysis, only the C-IBS subjects were identified. They were then evaluated for global improvement, abdominal pain, and constipation severity. The ability of neomycin to eliminate methane and its associated improvement in constipation was also determined. One hundred eleven subjects meeting Rome I criteria for IBS were included in the study. Thirty-nine of these had C-IBS. Of these, 20 received placebo and 19 received neomycin. With neomycin, a global improvement of 36.7±7.9% was seen, compared to 5.0±3.2% for placebo (P < .001) in the intention-to-treat analysis. Constipation was improved by 32.6±9.9% with neomycin compared to 18.7±7.2% for placebo (P = .26). Of the original 111 subjects, 12 demonstrated methane on breath test. All 12 of these patients were constipation predominant. In the methane producers receiving neomycin or placebo, improvement in constipation was significantly greater in those receiving neomycin (44.0±12.3%) compared to placebo (5.0±5.1%) (P < .05). Treatment with neomycin improves constipation in C-IBS. This improvement depends on the presence and elimination of methane on breath test.