Objective To assess the quality of after-hours clinical care as delivered by general practitioner (GP) cooperatives in the Netherlands. Methods A cross-sectional analysis was undertaken of patient health records of five GP cooperatives during 1 year. We used quality indicators derived from national guidelines for the appropriate prescription of pain medication and antibiotics, clinical performance in emergency cases and referral to medical specialists. Data were collected from electronic health records. Results We analysed 7660 patient contacts. Average adherence to the guidelines was 77%. The guidelines on referrals to medical specialists and prescription of pain medication had the highest adherence scores (92% and 90%, respectively). Prescribing antibiotics and treatment in emergency cases had the lowest scores (69% and 71%, respectively). Antibiotics were overprescribed in 42% of the cases and underprescribed in 21%. Conclusions In general, GPs adhered well to after-hours service national guidelines. There is room for improvement in care for people with acute illnesses and in the prescription of antibiotics.