Context Detecting oestrus to facilitate the optimal timing of artificial insemination is key to optimal reproductive performance in dairy cows. Aims The aims of the present study were to investigate the relationship between activity and milk oestradiol concentrations during oestrus. Methods Accelerometers (IceQubes, IceRobotics Ltd, UK) were used to monitor the activity (the number of steps per day) of 37 lactating Holstein–Friesian cows during the peri-oestrus period. Daily milk samples were analysed for oestradiol and progesterone concentrations. Key results An increase in activity sufficient to generate an oestrus alert (behavioural oestrus) from an activity sensor (GEA Rescounter II) was detected in 29 of 37 (78%) cows. Milk oestradiol increased from 2.0 ± 0.5 pg/mL 4 days before oestrus to 8.3 ± 1.1 pg/mL on the day of behavioural oestrus, then fell to 2.6 ± 0.6 pg/mL by 3 days afterwards. Similarly, activity also increased and peaked on the day of oestrus. However, in 8 of 37 (22%) cows, although milk oestradiol concentrations increased following the fall in progesterone, there was no significant increase in activity (silent oestrus). Milk oestradiol concentrations were positively correlated (r = 0.37; P = 0.03) with activity on the day of oestrus. Cows that subsequently became pregnant took a similar number of steps (2806 ± 282.3 vs 2850 ± 372.5 steps for PD+ vs PD− cows respectively) but had higher oestradiol concentrations (11.2 ± 2.06 vs 5.1 ± 0.51 pg/mL for PD+ vs PD− cows respectively) on the day of oestrus than did cows that did not become pregnant. Conclusions Increases in oestradiol concentrations above a threshold of approximately 10 pg/mL are associated with an increased activity, and higher oestral oestradiol concentrations are associated with a higher rate of pregnancy following insemination. Implications This study has demonstrated the significance of oestradiol concentration to the fertility of dairy cows. Thus, strategies to enhance oestradiol secretion may enhance fertility. Further, milk oestradiol measures may be developed as an aid to oestrus detection in lactating dairy cows, although further studies are required to determine whether on-farm assessment of milk oestradiol concentration can be developed to add to the repertoire of biomarkers of oestrus in lactating cattle.