Background Guidelines recommend that all health care professionals should be able to perform cardiopulmonary resuscitation (CPR), including the use of an automated external defibrillator. Theoretical knowledge of CPR is then necessary. The aim of this study was to investigate how much theoretical knowledge in CPR would increase among all categories of health care professionals lacking training in CPR, in an intervention hospital, after a systematic standardised training. Their results were compared with the staff at a control hospital with an ongoing annual CPR training programme. Methods Health care professionals at two hospitals, with a total of 3144 employees, answered a multiple-choice questionnaire before and after training in CPR. Bootstrapped chi-square tests and Fisher's exact test were used for the statistical analyses. Results In the intervention hospital, physicians had the highest knowledge pre-test, but other health care professionals including nurses and assistant nurses reached a relatively high level post-test. Improvement was inversely related to the level of previous knowledge and was thus most marked among other health care professionals and least marked among physicians. The staff at the control hospital had a significantly higher level of knowledge pre-test than the intervention hospital, whereas the opposite was found post-test. Conclusions Overall theoretical knowledge increased after systematic standardised training in CPR. The increase was more pronounced for those without previous training and for those staff categories with the least medical education.