It is unknown whether the ionic liquid markers CoEDTA and CrEDTA dissociate under ruminal conditions. Conditions in the rumen could reduce Co(III)- to Co(II)EDTA which is a less stable marker. As colored compounds, absorbances (ABS) of Co- and CrEDTA can be evaluated spectrophotometrically to determine if the molecules are intact, dissociated, or reduced. Wavelengths (λ) of peak ABS are 535, 465, and 560 nm for Co(III)EDTA, Co(II)EDTA, and Cr(III)EDTA, respectively, at pH 6.9. The objective of this study was to evaluate whether Co(III)- and Cr(III)EDTA markers dissociate or become reduced in vitro in simulated ruminal reducing conditions. In a complete block design with 2 replicate analytical runs and samples in duplicate within run, approximately 26 mg/L of Cr from Cr(III)EDTA or Co from Co(III)EDTA were incubated in Goering and Van Soest medium prepared without tryptone and with or without resazurin. In a 26 mL reaction volume were 20 mL of medium, 3 mL of Co(III)EDTA, or Cr(III)EDTA solutions, or water (reagent blanks), and 0, 0.25, 0.50, 0.75, or 1.00 mL reducing solution (RedSol) with water added to bring to 3.0 mL. Reduction status was determined with samples with resazurin, and ABS measures made on those without resazurin. Samples were maintained anaerobically. After incubation for 0.5 h at 39°C, ABS were read at λ 535, 465, and 560 nm. Mean reagent blank values were subtracted from Co- and CrEDTA data. Data were analyzed by marker with the MIXED procedure of SAS to detect differences in ABS at peak λ. The model included RedSol with analytical run as a random variable; contrasts were used to detect linear through quartic effects of RedSol. Resazurin color was clear for samples with > 0.25 mL RedSol, slightly pink with 0.25 mL, and remained blue with 0 mL. As RedSol increased, at 535 nm CoEDTA showed a cubic decline in ABS of 75% (P = 0.03), and a quadratic increase then decline (P < 0.01) at 465 nm. ABS of CrEDTA at 560 nm was unaffected by RedSol (linear P = 0.14). Co(III)EDTA apparently became reduced and dissociated to some extent under the in vitro conditions. This suggests that Co(III)EDTA is not a stable marker for use in studies under reducing ruminal conditions.