Effects of adaptation to L, M or a daily rotation of L and M (R) on in vitro fermentation were measured in a replicated in vitro experiment with a 4 × 4 factorial arrangement of treatments. Treatments were adaptation of ruminal fluid donor steers (0 or 200 mg steer−1 d−1 of L, M or R) and culture ionophore treatment (0 or 4 μg mL−1 of L, M or a 50:50 mixture of L and M). At 12 and 24 h, IVDMD was increased (P < 0.05) by adaptation to L compared with M, and decreased at 12 h (P < 0.05) by adaptation to R compared with the average of L and M adaptation. At 6 and 12 h, culture ionophore treatments increased (P < 0.10) IVDMD, compared with control. After 24 and 48 h of in vitro incubation, L adaptation increased (P < 0.01) acetate, decreased (P < 0.01) propionate, increased the acetate/propionate ratio (P < 0.01), and increased total VF A (P < 0.05), compared with M adaptation. Total VFA was increased (P < 0.10) for ionophore adaptation treatments compared with control at 48 h of incubation. Culture ionophore treatments decreased (P < 0.10) acetate and increased (P < 0.05) propionate at 24 and 48 h and decreased the acetate/propionate ratio (P < 0.10) at 24 h of incubation versus control cultures, with no measurable effects on total VFA. Adaptation treatments did not interact with culture treatments, suggesting that animals used as inoculum donors for in vitro experiments involving ionophores need not be adapted to an ionophore. Key words: In vitro, fermentation, ionophores, monensin, lasalocid