To examine the occurrence of p16 gene deletion and to analyze p16 expression on paraffin-embedded human pituitary adenoma specimens. Efforts were made to optimize the technical conditions for in situ PCR. Methods: In situ PCR techniques and inimuno-histochemistry were used. Results: Immunohistochemically, p16-positive tumor cells were observed in all cases with various proportions. The majority of the stromal cells and part of tumor cells was devoid of p16 immunostaining, but signal of in situ PCR for p16 gene, exon 2, was displayed in these cells. Conclusion: The results implied that p16 gene might not be deleted in these pituitary adenomas. It also indicated that in situ PCR, both direct and indirect methods, proved feasible and informative to the aim of DNA detection. It is critical to overcome non-specific amplification in direct in situ PCR by means of higher annealing temperature, fewer cycle, lower magnesium concentration and stringent washing. A target DNA-deleted sample as the negative control is extremely necessary. For the indirect method, the way to improve the sensitivity is to loosen the conditions for amplification and washing, so that more amplification products are subject to hybridization, and signal detection is facilitated.