The purpose of this study was to evaluate the effects of total replacement of soybean meal (SBM) with three alternative protein sources in the concentrate supplement on performance, feed efficiency, milk composition, and diurnal ingestive behavior of Holstein × Gyr cows under an intensive system of grazing on tropical pasture. Twelve cows with 512 ± 61kg of BW (mean ± standard deviation), 20.4 ± 4.14kg/d of milk yield, and 86 ± 40 days in milk were distributed in a triplicated 4 × 4 Latin square design. The treatments consisted of four concentrate supplements formulated with SBM (Control), cottonseed meal (CSM), peanut meal (PM), or sunflower meal (SFM). The animals were kept in intensive rotational grazing (n = 24 paddocks) on Tanzania grass (Panicum maximum syn. Megathyrsus maximus) pasture, with 1 day of occupation per paddock and a stocking rate of 4.3 AU/ha. The daily amount of concentrate supplement offered to the cows was 1kg DM of concentrate per 3kg of milk produced. Fat-corrected milk yield (FCM) for the SBM group (21.1kg/d) was similar to that of the other groups (21.5kg/d on average); however, milk fat content and milk urea nitrogen were lower in the SBM group (36g/kg and 12.6mg/dL) than in the PM group (38.4g/kg and 14.2mg/dL). The protein source did not affect the intakes of DM (17.14kg/d on average) and forage (9.66kg DM/d on average). However, cows fed CSM had a lower concentrate supplement intake compared to those fed PM (7.11 vs. 7.78kg DM/d). The lowest and the highest ether extract intake levels were observed in cows fed with SBM (0.31kg/d) and CSM (0.39kg/d). No effect of the protein source was detected on the time spent on grazing, rumination, and idleness, with mean values of 4.73,2.01, and 5.26 h within a 12 h observation period (06:00-18:00 h). Soybean meal can be replaced with CSM, PM, or SFM in the concentrate supplement without affecting the FCM, feed intake, feed efficiency, and diurnal ingestive behavior of lactating Holstein × Gyr cows in an intensive tropical grazing system. The replacement of SBM with PM is recommended since the latter promotes the best results in terms of milk fat content. All rights reserved, Elsevier.