Infertile men exhibit an aberrant protamine-1 (Prm1) to protamine-2 (Prm2) ratio at both the mRNA and protein level. We therefore investigated whether male infertility could be related to the amount of Prm1 and Prm2 mRNA by applying real time quantitative PCR following RNA extraction from routinely Bouin-fixed and paraffin-embedded testicular biopsies. Samples (n = 51) were normalized to the same amount and similar size of tissue sections. The threshold cycle (C(T)) representing a measure of the initial number of mRNA copies was significantly (P < 0.001) higher for Prm1, but not Prm2, and thus the amount of Prm1 mRNA was lower in men with at least qualitatively normal spermatogenesis (Prm1: 29.88 +/- 2.99; Prm2: 34.28 +/- 2.26) and impaired spermatogenesis (Prm1: 31.89 +/- 2.54; Prm2: 35.59 +/- 2.09) compared with men with obstructive azoospermia and quantitatively normal spermatogenesis (Prm1: 29.04 +/- 1.02; Prm2: 34.91 +/- 1.40). In addition, the Prm1 - Prm2 C(T) difference (deltaC(T)) was significantly (P < 0.001) decreased in these two groups. A negative correlation (r = -0.504; P < 0.001) was demonstrated between the score for efficiency of spermatogenesis and the C(T) for Prm1. These data suggest that the decreasing amount of Prm1 and, as a consequence, the aberrant Prm1:Prm2 mRNA ratio plays an important role for male infertility and may serve as a possible predictive factor for the outcome of ICSI.