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Purpose In order to introduce the concept of diagnostic reference levels (DRLs) in the national nuclear medicine practice a survey was proposed and completed through all nuclear medicine departments in Croatia. An additional aim was to increase the awareness of importance and full implementation of a comprehensive quality program that includes devices used in the nuclear medicine chain. Methods Data were collected for more than 30 nuclear medicine single photon emission procedures. National DRLs (NDRLs) as administered activity and also as administered activity per unit mass were calculated in accordance to International Commission on Radiological Protection (ICRP) recommendations. Additionally, effective doses were estimated using conversion factors published by the ICRP. Results NDRLs for nuclear medicine single photon emission procedures were proposed. For procedures performed in only one department typical values were presented as reference. Effective doses related to applied radiopharmaceuticals were calculated to estimate radiation risk related to respective nuclear medicine procedure in more detail. Conclusion This work presents results of the first national survey on DRLs of nuclear medicine single photon emission procedures and proposes reliable NDRLs that represent an actual status of nuclear medicine practice in Croatia. Results have motivated departments to introduce and set their own typical values to be used, as one of the tools, for further optimization process. One of the drawbacks of the DRL concept in nuclear medicine is the lack of the image quality parameters involved. For this reason, a quantity that considers both radiation protection and image quality should be introduced.