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Background  The relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization.    Results  Using bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of  Drosophila melanogaster , and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between  D. melanogaster  and  Drosophila pseudoobscura , their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data.    Conclusions  The combination of these analyses led us to postulate the existence of at least 401 functional smORFs in  Drosophila , with the possibility that as many as 4,561 such functional smORFs may exist.

Hundreds of putatively functional small open reading frames in Drosophila