We investigated relationships between circulating levels of salivary testosterone and the fundamental and formant frequencies of male voices in a sample of forty healthy adult males, who recorded their voices and provided saliva samples at 9 am, 12 noon and 3 pm on a single day. The relationship between 2D:4D ratio as a putative biomarker of prenatal testosterone and vocal parameters was also explored. Results supported previous findings for a negative relationship between circulating levels of testosterone and fundamental frequency, with higher testosterone indicating lower fundamental frequency, although the magnitude of the relationship was larger than previously observed. Some limited evidence for a relationship between circulating testosterone and formant dispersion is also found, although this did not reach significance. Diurnal variation in testosterone and fundamental frequency, but not formant dispersion was reported, together with a trend towards an association between the fall in testosterone and the rise in fundamental frequency. Finally, there was no relationship between 2D:4D and the vocal parameters. It is thought that male voices may have deepened over the course of evolution in order to signal dominance and/or to increase the speaker's attractiveness. Findings confirm that vocal frequencies may provide an honest signal of the speaker's hormonal quality.