This study was conducted to document the development of populations of lactic acid bacteria (LAB) and Lactobacillus buchneri in alfalfa silage treated with various inoculants. Wilted and chopped alfalfa (45% dry matter) was treated with 1) distilled water (untreated, U), 2) Lactobacillus buchneri 40788 (4 x 10(5) cfu/g; LB), or 3) L. buchneri 40788 (4 x 10(5) cfu/g) and Pediococcus pentosaceus (1 x 10(5) cfu/g; LBPP). Forages were packed into triplicate vacuum-sealed, nylon-polyethylene bags per treatment, and ensiled for 2, 5, 45, 90, and 180 d. Viable (cfu) LAB in forage and silage were quantified by traditional plating on selective agar, and numbers of L. buchneri (cfu-equivalent, cfu-E) were quantified by real-time quantitative PCR. Fresh, untreated forage had 5.52 log cfu of LAB/g and 3.79 log cfu-E of L. buchneri/g. After 2 d of ensiling, numbers of LAB increased to >8 log cfu/g in all silages. In contrast, numbers of L. buchneri in U remained below 4 log cfu-E/g but reached approximately 7 log cfu-E/g in LB and LBPP. From d 5 onward, numbers of L. buchneri in U remained below 6 log cfu-E/g but approached 9 log cfu-E/g in LB and LBPP. The pH was lower in LBPP compared with U and LB after 2 and 5 d of ensiling, but pH was lower for U compared with LB and LBPP thereafter. Treatments LB and LBPP had more acetic acid than U at 45 d of ensiling, which coincided with detectable amounts of 1,2 propanediol. Inoculation with LBPP resulted in silage with the highest concentration of 1,2 propanediol after 180 d of ensiling. From d 45 onward, LB and LBPP silages had lower concentrations of residual water-soluble carbohydrates but had higher concentrations of ammonia-N than U. In conclusion, epiphytic L. buchneri can be detected in alfalfa but this population is unable to lead the silage fermentation. In contrast, when L. buchneri was added to silage as an inoculant, the numbers of L. buchneri (cfu-E) increased markedly but did not dictate fermentation until 45 d of ensiling. These findings help to explain why the response (in increased acetic acid) from the addition of L. buchneri in silages is not immediate.