Two experiments were conducted to determine: 1) the impact of strained rumen fluid (SRF) alone or SRF with particle-associated microorganisms (PAO) included and dilution on in vitro dry matter digestibility (DMD) and 2) the impact of trace mineral (TM) source on in vitro fermentation characteristics and TM solubility under simulated abomasal and intestinal conditions. In Experiment 1, three cannulated steers were adapted to a diet formulated to meet the nutrient requirements for lactating dairy cows. Strained RF was obtained by straining rumen content through 2 layers of cheesecloth. Half of the remaining digesta was washed with McDougall's buffer and filtered through 2 layers of cheesecloth to obtain PAO. Both SRF and PAO were filtered again through 8 layers of cheesecloth. Strained RF was mixed with either McDougall's buffer (SRF) or PAO (SRF+PAO) at a ratio of 1:2 or 1:4 and incubated at 39°C for 12h using the ground basal diet as the substrate. Digestibility of DM was greater in digestion tubes containing SRF and SRF+PAO at a 1:2 ratio. In Experiment 2, eight steers fitted with ruminal cannula were blocked by body weight and assigned to one of two treatment groups. Treatments consisted of 10mg Cu, 40mg Mn, and 60mg Zn/kg DM from either: 1) sulfate (STM) or 2) hydroxychloride (HTM) sources. Steers were housed in individual pens and fed the same diet as described in experiment 1. Dietary TM treatments were mixed with dried distillers grains and mixed in the diet, by hand, immediately after basal diet delivery. Dietary treatments were fed for 14 d. On day 15, SRF+PAO was collected from each steer (STM-RF and HTM-RF) and used in a series of in vitro crossover experiments. In vitro substrates (S) used were the ground diets consumed by the animals on each treatment (STM-S and HTM-S). Incubations containing HTM-S had greater (P < 0.01) total VFA concentration and propionic acid molar proportions, but lesser (P < 0.01) acetic acid molar proportions than STM-S. Rumen fluid from steers supplemented with HTM had a greater (P < 0.03) total VFA than STM-RF at 24h post incubation. After 12h post incubation, the molar proportion of propionic acid in HTM-RF was lesser (P = 0.04) than STM-RF. After simulated abomasal digestion, soluble Mn concentration in HTM-S was greater (P < 0.01) than STM-S. These data indicate that the source of trace minerals can influence in vitro rumen fermentation characteristics and Mn solubility under simulated abomasal conditions.