Objectives: Several studies have shown that exposure to antineoplastic drugs can cause toxic effects on reproductive health as well as carcinogenic effects. Numerous studies have corroborated that hospital workers are exposed to these drugs. This study focused on trends in exposure to antineoplastic drugs since the introduction of guidelines in The Netherlands. Methods: Data from three cross-sectional exposure surveys conducted in The Netherlands were pooled to examine trends in occupational exposure to cyclophosphamide. Nurses' 24 h urine samples were analyzed in separate fractions, surface contamination was determined and gloves used during preparation or while handling patient urine were collected. The difference in detectable urine samples between 1997 and 2000 was determined by a generalized estimating equations (GEE) binomial regression model. Mixed models were used to study the time trend in surface and glove contamination levels. Results: The percentage of nurses' urine samples with detectable cyclophosphamide had decreased 4-fold between 1997 and 2000. Median cyclophosphamide levels in the positive urine samples were 3-fold lower in 2000 than in 1997. Surface and glove contamination had statistically significantly decreased between 1997 and more recent years. Conclusions: Nurses working at outpatient clinics or oncology wards are still being exposed to cyclophosphamide, but their exposure decreased considerably between 1997 and 2000, presumably due to the introduction of detailed guidelines and regulations in The Netherlands, the subsequent increased use of LuerLock connections and infusion systems prefilled with saline, and growing hazard awareness of nurses working with antineoplastic drugs. © The Author 2007. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.