The aim of this study was to compare plasma progesterone (P4) concentrations in nonlactating, multiparous Holstein cows (n = 24) treated with 2 types of intravaginal implants containing either 1.0 or 1.9 g of P4 either at the first use or during reuse of the implants after sanitizing the implant by autoclave or chemical disinfection. In a completely randomized design with a 2 × 3 factorial arrangement and 2 replicates, every cow underwent 2 of 6 treatments. Two sources of P4 [controlled internal drug release (1.9 g of P4) from Zoetis (São Paulo, Brazil), and Sincrogest (1.0 g of P4) from Ourofino (Cravinhos, Brazil)] and 3 types of processing, new (N), reused after autoclave (RA), and reused after chemical disinfection (RC), were used. After inducing luteolysis to avoid endogenous circulating P4, the cows were randomized in 1 of 6 treatments (1.9 g of N, 1.9 g of RA, 1.9 g of RC, 1.0 g of N, 1.0 g of RA, and 1.0 g RC). Cows were treated with the implants for 8 d and during this period blood samples were collected at 0, 2, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h. Statistical analyses were performed using Proc-Mixed and the mean ± standard error of the mean P4 concentrations were calculated using the Proc-Means procedures of SAS 9.4 (SAS Institute Inc., Cary, NC). No interaction between treatments was observed. Comparing types of implant, average P4 concentrations during treatments were greater for 1.9 g than 1.0 g (1.46 vs. 1.14 ± 0.04 ng/mL). When types of processing were compared, average P4 concentrations did not differ between autoclaved and new inserts (1.46 vs. 1.37 ± 0.05 ng/mL; respectively), but both were greater than chemically disinfected implants (1.09 ± 0.04 ng/mL). Within 1.9-g P4 inserts, P4 concentrations from autoclaved implants were greater than new, which were greater than chemically disinfected (1.67 ± 0.06 vs. 1.49 ± 0.07 vs. 1.21 ± 0.05 ng/mL; respectively). For 1.0-g P4 implants, P4 concentrations from autoclaved did not differ from new, but both were greater than chemically disinfected (1.20 ± 0.08 vs. 1.24 ± 0.06 vs. 0.97 ± 0.05 ng/mL; respectively). In conclusion, the mean plasma P4 concentration in nonlactating Holstein cows was greater for 1.9 than 1.0 g of P4 and regardless of the type of implant, the autoclaving process provided greater circulating P4 in relation to chemical disinfection, and similar or greater P4 concentrations compared with a new implant.