Background: Protamines are small basic proteins that condense the DNA in mature spermatozoa. Typical protamines are of simple composition and very arginine-rich, usually in the range of 60-80%. Arginine residues are distributed in a number of stretches separated by neutral amino acids. We have used Fourier transform infrared spectroscopy (FTIR) to gain access for the first time to the secondary structure of protamines in sperm nuclei. This technique is particularly well suited to the study of DNA-bound protamine in whole nuclei since it is not affected by turbidity. Results: We show that DNA -bound salmon (salmine) and squid protamines contain -helix, -turns and a proportion of other structures not stabilized by intramolecular hydrogen bonding. No -sheet was observed. In salmine, the -helix amounted to ∼20%, while in squid protamine it reached ∼40%. In contrast, the structure not stabilized by intermolecular hydrogen bonding was more abundant in salmine (∼40%) than in squid protamine (∼20%). Both protamines contained ∼40% -turns. The different helical potential of salmine and squid protamine was confirmed by structure predictions and CD in the presence of trifluoroethanol. Conclusion: DNA-bound protamine in sperm nuclei contains large amounts of defined secondary structure stabilized by intramolecular hydrogen bonding. Both salmine and squid protamine contain similar amounts of -turns, but differ in the proportions of -helix and non-hydrogen bonded conformations. In spite of the large differences in the proportions of secondary structure motifs between salmon and squid protamines, they appear to be equally efficient in promoting tight hexagonal packing of the DNA molecules in sperm nuclei. © 2011 Roque et al; licensee BioMed Central Ltd.