Combined pituitary hormone deficiency is characterized by the impaired production of pituitary hormones, commonly including growth hormone. The pathomechanism of the childhood-onset form of this disorder may involve germline mutations of genes encoding pituitary transcription factors, of which PROP1 gene mutations have been studied most extensively. However, controversy exists about the significance of PROP1 gene mutations, as both low and high frequencies have been reported in these patients. Because the different results may be related to differences in patient populations and/or the variability of clinical phenotypes, we performed the present study to examine the prevalence and spectrum of PROP1 gene mutations in 35 patients with non-acquired childhood-onset growth hormone deficiency combined with at least one other anterior pituitary hormone deficiency. Genetic testing indicated the presence of disease-causing mutations in exons 2 and 3 of the PROP1 gene in 15 patients (43% of all patients; homozygous mutations in 10 patients and compound heterozygous mutations in 5 patients). Comparison of clinical data of patients with and without PROP1 gene mutations failed to show significant differences, except an earlier growth retardation detected in patients with PROP1 gene mutations. In one patient with PROP1 gene mutation, radiologic imaging showed an enlargement of the anterior lobe of the pituitary, whereas the other patients had hypoplastic or normal pituitary gland. All patients with PROP1 gene mutations had normal posterior pituitary lobe by radiologic imaging. These results indicate that using our inclusion criteria for genetic testing, PROP1 gene mutations can be detected in a high proportion of Hungarian patients with non-acquired childhood-onset growth hormone deficiency combined with at least one other anterior pituitary hormone defect.