Method: From 2002 to 2012 we used Glyo-Fixx (Thermo Scientific, US), a glyoxal-based fixative, as formaldehyde substitute, analysing 54314 surgical pathology specimens and 205 autopsies. All samples were processed and stained routinely. Moreover we performed immunohistochemistry by an automated immunostainer and special stains with standard techniques, where needed. In tumoral cases we fixed 1 cm3 sample in neutral buffered formalin and compared the results with Gyo-Fixx fixation. Results: Glyo-Fixx fixation needed less hours than formaldehyde and not harden the tissues. On gross examination of the adipose tissue, lymph nodes were easily found, due to a more marked whitish appearance. Morphologically we have not found differences between formalin and Glyo-Fixx, except for the eosinophils degranulation. Special stains resulted similar to those with formaldehyde fixations. Almost all antibodies not required pretreatment, but needed adjusts in the standardized protocol for formaldehyde. Conclusion: Our experience demonstrated Glyo-Fixx is a good non-toxic alternative to formaldehyde in routine pathology, capable to preserve morphology and protein integrity of the tissues.