The objective of this experiment was to investigate the effect of trace mineral (TM) source on Cu, Mn, and Zn rumen solubility and in vitro release from the insoluble portion of rumen digesta using dialysis. Eight cross-bred steers (BW 718.9 ± 64.9 kg), fitted with ruminal cannulas, received TM treatments (n = 4/treatment) as a bolus-dose directly into the rumen. Treatments consisted of (1) sulfate TM (STM): 20 mg Cu/kg DM from CuSO₄; 40 mg Mn/kg DM from MnSO₄; 60 mg Zn/kg DM from ZnSO₄, and (2) Hydroxychloride TM (HTM): 20 mg Cu/kg DM from tribasic CuCl; 40 mg Mn/kg DM from Mn hydroxychloride; 60 mg Zn/kg DM from Zn hydroxychloride. Rumen samples were obtained at 2 h intervals starting at −4, and ending at 24 h post dosing. Once collected, samples were centrifuged and supernatant and pellet fractions were analyzed for TM. For dialysis, pellets from the 0, 6, and 12 h sampling times were dialyzed against one of the following chelating buffers: 1) 0.01M ethylenediaminetetraacetate in 0.05 M Tris (Tris-EDTA; strong chelator) or 2) 0.01M L- histidine hydrochloride in 0.05M Tris (weak chelator). Data were analyzed as a randomized block design with repeated measures. A treatment x time interaction (P < 0.05) existed for rumen soluble Cu and Zn concentrations. Rumen soluble Cu concentrations were lesser (P < 0.05) from 2 to 12 h post dosing (mean difference = 0.18 vs. 0.44 ± 0.06 mg Cu/L) and rumen soluble Zn concentrations were lesser (P < 0.04) from 2 to 10 h post dosing (mean difference = 0.75 vs. 1.29 ± 0.13 mg Zn/L) for steers receiving HTM compared with steers receiving STM. Dialysis against Tris-EDTA resulted in a greater (P < 0.05) percent release of Cu (78.2 vs. 30.2 ± 20.3) from samples obtained 12 h post dosing and a greater (P < 0.04) percent release of Zn from samples obtained at 6 (89.3 vs. 41.2 ± 14.8) and 12 (77.8 vs. 31.3 ± 18.3) h post dosing in HTM compared with STM steers. These data indicate that rumen solubility of Cu and Zn is reduced for HTM compared with STM sources and that Cu and Zn are less tightly bound in the insoluble rumen fraction of steers fed HTM compared with STM sources.