Background <p>The model rumen-dominant ureolytic bacterium <i>P. penneri</i> S99 exhibits high urease activity. It was cultivated using ammonia, urea, amino acids, or their combination as nitrogen sources. To identify differences in gene expression, the transcript abundances of various genes involved in nitrogen metabolism were analyzed by harvesting mRNA from cells during the exponential growth phases on different nitrogen sources.</p> Results <p><i>P. penneri</i> S99 can utilize ammonia, urea, or amino acids as the sole nitrogen sources for growth and shows a preference for utilizing urea. It exhibits similar growth rates and maximum biomass on ammonia and urea, but showed higher growth rates and maximum biomass on amino acids. Transcriptome sequencing analysis revealed different transcription patterns in response to different nitrogen sources. The urease gene expression was detected in all three different nitrogen sources, and complete hydrolysis of urea was also observed when other nitrogen sources were added to the medium containing urea. The regulation of urease in <i>P. penneri</i> S99 was characterized by constitutive expression, not by urea. The growth of <i>P. penneri</i> S99 on ammonia, ammonium acid, and urea was similar, with the only observed difference being an increase in urease transcript abundance. </p> Conclusions <p>The transcription patterns of nitrogen metabolism genes offer insights into how nitrogen is utilized in the rumen.</p>