The aim of this study was to measure the NO level in boar semen held in a liquid state and to determine its putative relation to spermatozoa motility, plasma membrane integrity, mitochondrial membrane potential and ATP content. Generally, the percentage of spermatozoa which generated nitric oxide gradually increased, while NO level in the surrounding medium declined during the liquid preservation. NO generation in semen preserved in BTS was higher as compared to those in Androhep®Plus. We demonstrated the positive correlation between the NO level in fresh spermatozoa and their quality. We also showed negative correlation between nitric oxide level in spermatozoa preserved in BTS and sperm cells motility as well as plasma membrane integrity. Results obtained in this study confirm that NO may affect sperm physiology in a dualistic manner.