In this study, we investigated the ability of the fungus Neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine RNase A and the endogenous RNase N-1. A set of expression vectors was constructed, each consisting of an RNase A open reading frame under the control of a specific promoter and each with a specific terminator. N. crassa transformants were analyzed at the transcriptional and protein levels. Irrespective of the promoter used, all transformants showed an RNase A-specific transcript in northern hybridization, but transcriptional strengths differed significantly. The strongest transcription was detected in transformants under the control of the cfp promoter. Western blot analysis and ELISA assays of selected transformants showed an effective secretion up to 356 ng/mL of recombinant RNase A protein. However, the highest ribonuclease activity could be detected in transformants carrying the endogenous RNase N-1 under the control of the ccg1 promoter. Expression and secretion of RNase N-1 thus represent an alternative to recombinant expression of RNase A protein. In conclusion, we have created a viable expression system for expression of homologous and heterologous proteins in N. crassa.