The present study aims to examine the influence of evening and night shift work, compared to day shift work, on melatonin secretion in nurses in a field setting. Effects were examined during a workday and during a day off. Both fixed schedules and mixed or rotating schedules were studied. In total, 170 nurses were studied: 89 nurses worked fixed schedules, 27 nurses worked the day shift, 12 nurses worked the evening shift, 50 nurses worked the night shift, and 82 nurses worked mixed schedules, with data collected during a day (n = 17), evening (n = 14), or night shift (n = 50). All spot urine samples were collected during 24 h from the participants on a work day and on a day off and were analyzed for 6-sulphatoxymelatonin. On the day of urine sampling, participants filled in the Karolinska Sleep Diary. Additional information was collected through a telephone interview. Data were analyzed using a mixed procedure with autoregressive covariance structure. The present study showed that shift work affected the concentrations of 6-sulphatoxymelatonin in the short term by lower excretion in urine from nurses working the night compared to day shift on a workday and on a day off as well. No significant differences were observed between a workday and a day off when doing day and evening shifts, irrespective of mixed and fixed schedules. Sleep length was reduced workdays (from 6.1-6.8 h) among all nurses, compared to days off (from 7.8-8.7 h).