OBJECTIVE: To evaluate the clinical application of national guidelines for neonatal nursing. DESIGN: Questionnaire survey. SETTING: Thirty-nine neonatal care units in Sweden. STUDY PARTICIPANTS: Thirty-five of 39 nurse managers at all Swedish neonatal care units. INTERVENTION: Thirteen clinical guidelines for neonatal nursing care were presented in 1997. Recommendations on evidence-based nursing care and auditing measures were given. Most neonatal units in Sweden participated in the guideline development. MAIN OUTCOME MEASURES: Extent of guideline application, ways in which the guidelines were used and perceived usefulness. RESULTS: The guidelines were applied to different extents in 30 of the 35 units. Almost all the guidelines were applied, especially those covering general nursing care. In total, 72 Quality Improvement (QI) projects were reported, of which 51 concerned specific topics covered in the guidelines. Twenty units applied the guidelines as a starting point for QI. Four units evaluated nursing practice against the guidelines. Four factors [Dynamic Standard Setting System (DySSSy) as the QI method, > or = 4 years of practice as nurse manager, experience of nursing research, and good staff resources] were closely related to a more extensive application of the guidelines. Units with both a nurse manager and an assistant nurse manager were more likely to have used the guidelines as the basis for changing clinical practice. CONCLUSIONS: The guidelines were successfully disseminated and diffused, but practitioner involvement in guideline development did not guarantee implementation. Downsizing, leadership and facilitation seemed to be crucial factors when getting evidence into practice. Limited occurrence of evaluations of clinical practice against guideline recommendations suggests a need for valid and user-friendly measures.