The aim of this study was to observe the development of exploratory behaviour in growing broilers, i.e. their reaction to a novel environment (habituation) during the post-incubation period. Twelve males and twelve females of ROSS 308 line were kept from hatching until 42 days of age in separate pens, under identical husbandry conditions. To study their behaviour, we used the open-field test (OFT). Individual 10-min tests were initiated at the age of 3 days, and were repeated at weekly intervals, i.e. a total of 6 tests were carried out. The results were evaluated using analysis of variance (ANOVA) for repeated measures and Tukey post-hoc test. Broilers of both sexes were most active during the first OFT on day 3 after hatching. In male broilers, we found a significant decrease in all exploratory activities between the first and 2 nd OFT; duration of their horizontal locomotor activity was reduced from 104 ± 51.86 s to 3.50 ± 3.50 s (p < 0.05), duration of visual orientation decreased from 327.50 ± 55.94 s to 125.00 ± 31.94 s (p < 0.001), and duration of vocalizations decreased from 412.50 ± 63.12 s to 90.00 ± 34.25 s (p < 0.001). In females, a significant decrease was observed betwen the first and 2 nd OFT only in duration of horizontal locomotor activity that decreased from 234.25 ± 52.07 s to 14.25 ± 8.36 s (p < 0.001) and between the first and 3 rd OFT in duration of vocalization (decreasing from 430.00 ± 66.30 s in 1 st OFT to 60.00 ± 23.35 s; p < 0.001). On the other hand, a significant increase in duration of comfort behaviour was noted between the first and second OFT in both sexes (in males from 95.00 ± 40.76 s to 462.50 ± 38.81 s; p < 0.001; in females from 85.00 ± 31.73 s to 437.50 ± 67.74 s; p < 0.001). Changes in defecation numbers were not significant in either sex. Our findings show that the most profound changes in behaviour of broiler chickens occurred between the first and second OFT, that male broilers used visual orientation in the open field arena more than females in the first test, they vocalized less in the second test, and they seem to have habituated to the novel environment earlier than females.