Megasphaera elsdenii (Me) is a lactate-utilizing bacterium whose ruminal abundance has been shown to be greatly elevated during milk fat depression (MFD). To further examine this association, a total of 23 cannulated multiparous Holstein cows were examined in 3 studies in which strains of Me were directly dosed into the rumen (~2 × 10¹² cells/dose); control cows were dosed with sterile lactate-free culture medium. Cows were fed a TMR (30% starch, dry matter (DM) basis) that contained primarily corn silage, alfalfa silage, finely ground high-moisture corn, supplemental protein, and corn oil (3 g/kg DM). Studies differed in stage of lactation of the cows (middle or late), dosing events (single dose, or 4 doses over a 5 d period), timing of dose (pre-feed or 3 h postfeed), and Me strain (lab strain YI9 or 3 strains isolated from cows in the same herd). DM intake, milk yield and composition were measured from 5 d to 0 d before dosing and 1 to 7 d after first dosing, plus later time points that varied by experiment. None of these measures were affected by dosing (P > 0.05). Me was quantified in the liquid phase of ruminal contents by polymerase chain reaction (PCR) with relative quantification (Me 16S rRNA gene copy number as a percentage of total bacterial 16S rRNA gene copies). Neither the Me-dosed or control cows displayed MFD after dosing, and in almost all cases Me populations returned to low baseline levels (<0.02% of 16S rRNA gene copy number) within 24 h of dosing. This rapid decline in Me also occurred in several cows that were dosed with a strain that had been isolated from that particular cow during a previous bout of MFD. Ruminal pH, total mM volatile fatty acids (VFA) and mM lactate did not differ between dosed and control cows (P > 0.05), although acetate:propionate ratio declined (P < 0.01) in both groups, and butyrate increased after dosing with Me (P < 0.05). The results confirm that establishing exogenously added bacterial strains in the rumen is difficult, even for strains previously isolated from the recipient cow. The potential role of Me as an agent of MFD remains unclear in the absence of successful establishment of the dosed strains.